Use of R-enantiomer of N-propargyl-1-aminoindan, salts, and compositions thereof

ABSTRACT

The subject invention provides R(+)-N-propargyl-1-aminoindan and pharmaceutically acceptable salts thereof, as well as pharmaceutical compositions containing same. The subject invention also provides methods of treating a subject afflicted with Parkinson&#39;s disease, a memory disorder, dementia, depression, hyperactive syndrome, an effective illness, a neurodegenerative disease, a neurotoxic injury, stroke, brain ischemia, a head trauma injury, a spinal trauma injury, neurotrauma, schizophrenia, an attention deficit disorder, multiple sclerosis, or withdrawal symptoms, using R(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable salt of the subject invention. The subject invention further provides a method of preventing nerve damage in a subject. Finally, the subject invention provides methods of preparing R(+)-N-propargyl-1-aminoindan, a salt thereof, and racemic N-propargyl-1-aminoindan.

[0001] Throughout this application, various references are referred to.Disclosures of these publications in their entireties are herebyincorporated by reference into this application to more fully describethe state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

[0002] I.

[0003] The subject invention is in the field of selective irreversibleinhibitors of the enzyme monoamine oxidase (hereinafter MAO) andprovides the R(+) enantiomer of N-propargyl-1-aminoindan (also referredto herein as PAI) which is a selective irreversible inhibitor of theB-form of monoamine oxidase enzyme (hereinafter MAO-B). The subjectinvention also provides pharmaceutical compositions containing R(+)PAIwhich are particularly useful for the treatment of Parkinson's disease,a memory disorder, dementia, depression, hyperactive syndrome, anaffective illness, a neurodegenerative disease, a neurotoxic injury,stroke, brain ischemia, a head trauma injury, a spinal trauma injury,neurotrauma, schizophrenia, an attention deficit disorder, multiplesclerosis, and withdrawal symptoms.

[0004] II.

[0005] Parkinson's disease is widely considered to be the result ofdegradation of the pre-synaptic dopaminergic neurons in the brain, witha subsequent decrease in the amount of the neurotransmitter dopaminebeing released. Inadequate dopamine release, therefore, leads to theonset of disturbances of voluntary muscle control, which disturbancesare symptomatic of Parkinson's disease.

[0006] Various methods of treating Parkinson's disease have beenestablished and are currently in widespread use, including, for example,the administration of L-DOPA together with a decarboxylase inhibitorsuch as L-carbidopa or benserazide. The decarboxylase inhibitor protectsthe L-DOPA molecule from peripheral decarboxylation and thus ensuresL-DOPA uptake by the remaining dopaminergic neurons in the striatum ofthe brain. Here, the L-DOPA is converted into dopamine resulting inincreased levels of dopamine in these neurons. In response tophysiological impulses, these neurons are therefore capable of releasinglarger amounts of dopamine at levels which approximate the normalrequired levels. L-DOPA treatment thus alleviates the symptoms of thedisease and contributes to the well-being of the patient.

[0007] However, L-DOPA treatment has its drawbacks, the main one beingthat its effectiveness is optimal only during the first few years oftreatment. After this period, the clinical response diminishes and isaccompanied by adverse side effects which include dyskinesia,fluctuation in efficacy throughout the day (“on-off effect”) andpsychiatric symptoms such as confusional states, paranoia, andhallucinations. This decrease in the effect of L-DOPA treatment isattributed to a number of factors, including the natural progression ofthe disease, alteration in dopamine receptors as a consequence ofincreased dopamine production or increased levels of dopaminemetabolites, and pharmacokinetic problems of L-DOPA absorption (reviewedby Youdim, et al., Progress in Medicinal Chemistry, 21, 138-167 (1984)).

[0008] In order to overcome the drawbacks of L-DOPA treatment, varioustreatments have been devised in which L-DOPA is combined with MAOinhibitors with the aim of reducing the metabolic breakdown of newlyformed dopamine (see for example, Chiese, P., U.S. Pat. No. 4,826,875,issued May 2, 1989).

[0009] MAO exists in two forms known as MAO-A and MAO-B which areselective for different substrates and inhibitors. For example, MAO-Bmore efficiently metabolizes substrates such as 2-phenylethylamine, andis selectively and irreversibly inhibited by (−)-deprenyl as describedbelow.

[0010] It should be noted, however, that treatments combining L-DOPAwith an inhibitor of both MAO-A and MAO-B are undesirable, as they leadto adverse side effects related to an increased level of catecholaminesthroughout the neuraxis. Furthermore, complete inhibition of MAO is alsoundesirable as it potentiates the action of sympathomimetic amines suchas tyramine, leading to the so-called “cheese effect” (reviewed byYoudim et al., Handbook of Experimental Pharmacology, ed. byTrendelenburg and Weiner, Springer-Verlag, 90, ch. 3 (1988)). As MAO-Bwas shown to be the predominant form of MAO in the brain, selectiveinhibitors for this form are thus considered to be a possible tool forachieving a decrease in dopamine breakdown on the one hand, togetherwith a minimization of the systemic effects of total MAO inhibition onthe other.

[0011] Many inhibitors of MAO are chiral molecules. Although oneenantiomer often shows some stereoselectivity in relative potencytowards MAO-A and -B, a given enantiomeric configuration is not alwaysmore selective than its mirror image isomer in discriminating betweenMAO-A and MAO-B.

[0012] Table I lists the IC₅₀ (mmol/L) of enantiomeric pairs ofpropargyl amines in a rat brain preparation of MAO. These results showsmall differences in potency in MAO-B inhibition between the R and Senantiomers. (B. Hazelhoff, et al., Naunyn-Schmeideberg's Arch.Pharmacol., 330, 50 (1985)). Both enantiomers are selective for MAO-B.In 1967, Magyar, et al. reported that R-(−)-deprenyl is 500 times morepotent than the S-(+) enantiomer in inhibiting the oxidative deaminationof tyramine by rat brain homogenate. (K. Magyar, et al., Act. Physiol.Acad. Sci., Hung., 32, 377 (1967)).

[0013] In rat liver homogenate, R-deprenyl is only 15 times as potent asthe S enantiomer. In other pharmacological activity assays, such as forthe inhibition of tyramine uptake, deprenyl shows differentstereoselectivities. The S form is in certain cases the more potentepimer. (J. Knoll and K. Macyar, Advances in BiochemicalPsychopharmacology, 5, 393 (1972)).

[0014] N-Methyl-N-propargyl-1-aminotetralin (2-MPAT) is a closestructural analogue of deprenyl. The absolute stereo-chemistry of 2-MPAThas not been assigned. However, the (+) isomer is selective for MAO-Band the (−) isomer is selective for MAO-A. The difference in potencybetween the 2-MPAT enantiomers is less than 5-fold. (B. Hazelhoff, etal., id.). The enantiomers of N-propargyl-1-aminotetralin (1-PAT) arealso similar in activity. The lack of data in Table I showing clearstructure-activity relationships between isolated (+) or (−)-2-MPATmakes it impossible to predict the absolute stereochemistry thereof.

[0015] After extensive computer modeling, Polymeropoulos recentlypredicted that (R)-N-methyl-N-propargyl-1-aminoindan (R-1-MPAI) would bemore potent than (S) as a MAO-B inhibitor. (E. Polymeropoulos,Inhibitors of Monoamine Oxidase B, I. Szelenyi, ed., Birkhauser Verlag,p. 110 (1993)). However, experiments described show that R-1-MPAI is aslightly more potent inhibitor of MAO-B than S-1-MPAI, but is an evenmore potent inhibitor of MAO-A. Both the selectivity between MAO-A and-B and the relative potency of the R and S epimers are low. Thus,contrary to expectations in the art, 1-MPAI is useless as apharmaceutical agent.

[0016] The data presented below demonstrate that high selectivity forMAO of one enantiomer versus the other cannot be predicted. Thestructure of the MAO active site is not well enough understood to permitthe prediction of the relative potency or selectivity of any givencompound or pair or enantiomers thereof.

[0017] III.

[0018] Brain stroke is the third leading cause of death in the developedcountries. Survivors often suffer from neurological and motordisabilities. The majority of CNS strokes are regarded as localizedtissue anemia following obstruction of arterial blood flow which causesoxygen and glucose deprivation. Occlusion of the middle cerebral arteryin the rat (MCAO) is a common experimental procedure that is assumed torepresent stroke in humans. It has been proposed that the neurologicallesion caused by proximal occlusion of this artery in the ratcorresponds to a large focal cerebral infarct in humans (Yamori et al.,1976). This correspondence has been based on similarities betweencranial circulation in the two species. Other animal models of strokehave been described by Stefanovich (1983).

[0019] The histological changes described by Tamura et al. (1981) whowere the first to introduce the MCAO procedure, were commonly seen inthe cortex of the frontal (100%), sensimotor (75%) and auditory (75%)areas and to a lesser extent in the occipital lobe cortex (25%). Inaddition, damage was observed in the lateral segment of the caudatenucleus (100%), and only to a variable extent in its medial portion(38%). Concomitantly, the following disorders were reported in MCAOanimals: neurological deficits (Menzies et al., 1992), cognitivedisturbances (Yamamoto et al., 1988), brain edema (Young et al., 1993;Matsui et al., 1993; Saur et al., 1993), decreased cerebral blood flow(Teasdale et al., 1983), catecholamine fluctuations. (Cechetto et al.,1989). Any of these disorders might be indicative of the severity andextent of brain damage that follow MCAO in the rat. Conversely, a drugwith a potential to limit or abort a given disorder may be considered asa candidate for the treatment of stroke in humans. TABLE IA IC₅₀(mmol/L) Data for Rat Brain MAO Inhibition by Propargylamines RELATIVECOM- INHIBITION POTENCY POUND REF EPIMER A B A/B A +/− B 2-MPAI a + 14016 8.8 3 0.2 − 46 88 0.5 DEPRENYL a S 3600 16 120 R/S R 450 6 75 80 2.61-MPAI b S 70 50 1.4 23 5 R 3 10 0.3 1-PAT c S 3800 50 76 4 0.5 R 900 9010

[0020] One selective MAO-B inhibitor, (−)-deprenyl, has been extensivelystudied and used as a MAO-B inhibitor to augment L-DOPA treatment. Thistreatment with (−)-deprenyl is generally favorable and does not causethe “cheese effect” at doses causing nearly complete inhibition of MAO-B(Elsworth, et al., Psychopharmacology, 57, 33 (1978)). Furthermore, theaddition of (−)-deprenyl to a combination of L-DOPA and a decarboxylaseinhibitor administered to Parkinsons's patients leads to improvements inakinesia and overall functional capacity, as well as the elimination of“on-off” type fluctuations (reviewed by Birkmayer & Riederer in“Parkinson's Disease,” Springer-Verlag, pp. 138-149 (1983)). Thus,(−)-deprenyl (a) enhances and prolongs the effect of L-DOPA, and (b)does not increase the adverse effects of L-DOPA treatment.

[0021] However, (−)-deprenyl is not without its own adverse sideseffects, which include activation of pre-existing gastric ulcers andoccasional hypertensive episodes. Furthermore, (−)-deprenyl is anamphetamine derivative and is metabolized to amphetamine andmethamphetamines, which substances may lead to undesirable side effectssuch as increased heart rate (Simpson, Biochemical Pharmacology, 27,1951 (1978); Finberg, et al., in “Monoamine Oxidase Inhibitors—The Stateof the Art,” Youdim and Paykel, eds., Wiley, pp. 31-43 (1981)).

[0022] Other compounds have been described that are selectiveirreversible inhibitors of MAO-B but which are free of the undesirableeffects associated with (−)-deprenyl. One such compound, namelyN-propargyl-1-aminoindan.HCl (racemic PAI.HCl), was described in GB1,003,686 and GB 1,037,014 and U.S. Pat. No. 3,513,244, issued May 19,1970. Racemic PAI.HCl is a potent, selective, irreversible inhibitor ofMAO-B, is not metabolized to amphetamines, and does not give rise tounwanted sympathomimetic effects.

[0023] In comparative animal tests, racemic PAI was shown to haveconsiderable advantages over (−)-deprenyl. For example, racemic PAIproduces no significant tachycardia, does not increase blood pressure(effects produced by doses of 5 mg/kg of (−)-deprenyl), and does notlead to contraction of nictitating membrane or to an increase in heartrate at doses of up to 5 mg/kg (effects caused by (−)-deprenyl at dosesover 0.5 mg/kg). Furthermore, racemic PAI.HCl does not potentiate thecardiovascular effects of tyramine (Finberg, et al., in “Enzymes andNeurotransmitters in Mental Disease,” pp. 205-219 (1980), Usdin, et al.,Eds., Wiley, New York; Finberg, et al. (1981), in “Monoamine oxidaseInhibitors—The State of the Art,” ibid.; Finberg and Youdim, BritishJournal Pharmacol., 85, 451 (1985)).

[0024] One underlying object of this invention was to separate theracemic PAI. compounds and to obtain an enantiomer with MAO-B inhibitionactivity which would be free of any undesirable side effects associatedwith the other enantiomer.

[0025] Since deprenyl has a similar structure to PAI and it is knownthat the (−)-enantiomer of deprenyl, i.e. (−)-deprenyl, is considerablymore pharmaceutically active than the (+)-enantiomer, the (−) enantiomerof PAI would be expected to be the more active MAO-B inhibitor.

[0026] However, contrary to such expectations, upon resolution of theenantiomers, it was found that the (+)-PAI enantiomer is in fact theactive MAO-B inhibitor while the (−)-enantiomer shows extremely lowMAO-B inhibitory activity. Furthermore, the (+)-PAI enantiomer also hasa degree of selectivity for MAO-B inhibition surprisingly higher thanthat of the corresponding racemic form, and should thus have fewerundesirable side effects in the treatment of the indicated diseases thanwould the racemic mixture. These findings are based on both in vitro andin vivo experiments as discussed in greater detail infra.

[0027] It was subsequently shown that (+)-PAI has the R absoluteconfiguration. This finding was also surprising based on the expectedstructural similarity of (+)-PAI analogy with deprenyl and theamphetamines.

[0028] The high degree of stereoselectivity of pharmacological activitybetween R(+)-PAI and the S(−) enantiomer as discussed hereinbelow isalso remarkable. The compound R(+)-PAI is nearly four orders ofmagnitude more active than the S(−) enantiomer in MAO-B inhibition. Thisratio is significantly higher than that observed between the twodeprenyl enantiomers (Knoll and Magyar, Adv. Biochem. Psychopharmacol.,5, 393 (1972); Magyar, et al., Acta Physiol. Acad. Sci. Hung., 32, 377(1967)). Furthermore, in some physiological tests, (+)-deprenyl wasreported to have activity equal to or even higher than that of the (−)enantiomer (Tekes, et al., Pol. J. Pharmacol. Pharm., 40, 653 (1988)).

[0029] MPAI is a more potent inhibitor of MAO activity, but with lowerselectivity for MAO-B over A (Tipton, et al., Biochem. Pharmacol., 31,1250 (1982)). As only a small degree of difference in the relativeactivities of the two resolved enantiomers was surprisingly observedwith MPAI, the remarkable behavior of R(+)PAI is further emphasized (SeeTable 1B).

[0030] The subject invention also provides methods of using thepharmaceutically active PAI-enantiomer alone (without L-DOPA) fortreatment of Parkinson's disease, a memory disorder, dementia,depression, hyperactive syndrome, an affective illness, aneurodegenerative disease, a neurotoxic injury, brain ischemia, a headtrauma injury, a spinal trauma injury, schizophrenia, an attentiondeficit disorder, multiple sclerosis, or withdrawal symptoms (see reviewby Youdim, et al., in Handbook of Experimental Pharmacology,Trendelenberg and Wiener, eds., 90/I, ch. 3 (1988)).

[0031] The subject invention further provides a method of using thepharmaceutically active PAI-enantiomer alone for pre-treatment ofParkinson's disease. The subject invention also provides pharmaceuticalcompositions comprising R(+)PAI and synergistic agents such as levodopa.The use of such agents has been studied with respect to (−)-deprenylwhich was shown to be effective when administered alone to earlyParkinson's patients, and may also have a synergistic effect in thesepatients when administered together with α-tocopherol, a vitamin Ederivative (The Parkinson's Study Group, New England J. Med., 321(20),1364-1371 (1989)).

[0032] In addition to its usefulness in treating Parkinson's disease,(−)-deprenyl has also been shown to be useful in the treatment ofpatients with dementia of the Alzheimer type (DAT) (Tariot, et al.,Psychopharmacology, 91, 489-495 (1987)), and in the treatment ofdepression (Mendelewicz and Youdim, Brit. J. Psychiat. 142, 508-511(1983)). The R(+)PAI compound of this invention, and particularly themesylate salt thereof, has been shown to restore memory. R(+)PAI thushas potential for the treatment of mentory disorders, dementia,especially of the Alzheimer's type, and hyperactive syndrome inchildren.

[0033] Finally, the subject invention provides highly stable salts ofR(+)PAI with superior pharmaceutical properties. The mesylate salt isespecially stable, shows unexpectedly greater selectivity, and showssignificantly fewer side effects than do the corresponding racemicsalts.

SUMMARY OF THE INVENTION

[0034] The subject invention provides R(+)-N-propargyl-1-aminoindanhaving the structure:

[0035] The subject invention further provides a pharmaceuticallyacceptable salt of R(+)-N-propargyl-1-aminoindan.

[0036] The subject invention further provides a pharmaceuticalcomposition which comprises a therapeutically effective amount ofR(+)-N-propargyl-1-aminoindan or a pharmaceutically acceptable saltthereof and a pharmaceutically acceptable carrier.

[0037] The subject invention further provides a method of treating asubject afflicted with Parkinson's disease which comprises administeringto the subject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat Parkinson's disease in the subject.

[0038] The subject invention further provides a method of treating asubject afflicted with a memory disorder which comprises administeringto the subject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat the memory disorder in the subject.

[0039] The subject invention further provides a method of treating asubject afflicted with dementia which comprises administering to thesubject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat dementia in the subject. In one embodiment, thedementia is of the Alzheimer type (DAT).

[0040] The subject invention further provides a method of treating asubject afflicted with depression which comprises administering to thesubject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat depression in the subject.

[0041] The subject invention further provides a method of treating asubject afflicted with hyperactive syndrome which comprisesadministering to the subject an amount of R(+)-N-propargyl-1-aminoindanor the pharmaceutically acceptable salt thereof of the subject inventioneffective to treat hyperactive syndrome in the subject.

[0042] The subject invention further provides a method of treating asubject afflicted with an affective illness which comprisesadministering to the subject an amount of R(+)-N-propargyl-1-aminoindanor the pharmaceutically acceptable salt thereof of the subject inventioneffective to treat the affective illness in the subject.

[0043] The subject invention further provides a method of treating asubject afflicted with a neurodegenerative disease which comprisesadministering to the subject an amount of R(+)-N-propargyl-1-aminoindanor the pharmaceutically acceptable salt thereof of the subject inventioneffective to treat the neurodegenerative disease in the subject.

[0044] The subject invention further provides a method of treating asubject afflicted with a neurotoxic injury which comprises administeringto the subject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat the neurotoxic injury in the subject.

[0045] The subject invention further provides a method of treating asubject afflicted with brain ischemia which comprises administering tothe subject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat brain ischemia in the subject.

[0046] The subject invention further provides a method of treating asubject afflicted with a head trauma injury which comprisesadministering to the subject an amount of R(+)-N-propargyl-1-aminoindanor the pharmaceutically acceptable salt thereof of the subject inventioneffective to treat the head trauma injury in the subject.

[0047] The subject invention further provides a method of treating asubject afflicted with a spinal trauma injury which comprisesadministering to the subject an amount of R(+)-N-propargyl-1-aminoindanor the pharmaceutically acceptable salt thereof of the subject inventioneffective to treat the spinal trauma injury in the subject.

[0048] The subject invention further provides a method of treating asubject afflicted with schizophrenia which comprises administering tothe subject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat schizophrenia in the subject.

[0049] The subject invention further provides a method of treating asubject afflicted with an attention deficit disorder which comprisesadministering to the subject an amount of R(+)-N-propargyl-1-aminoindanor the pharmaceutically acceptable salt thereof of the subject inventioneffective to treat the attention deficit disorder in the subject.

[0050] The subject invention further provides a method of treating asubject afflicted with multiple sclerosis which comprises administeringto the subject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat multiple sclerosis in the subject.

[0051] The subject invention further provides a method of preventingnerve damage in a subject which comprises administering to the subjectan amount of R(+)-N-propargyl-1-aminoindan or the pharmaceuticallyacceptable salt thereof of the subject invention effective to preventnerve damage in the subject.

[0052] The subject invention further provides a method of treating asubject suffering from symptoms of withdrawal from an addictivesubstance which comprises administering to the subject an amount ofR(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable saltthereof of the subject invention effective to treat the symptoms ofwithdrawal in the subject.

[0053] The subject invention further provides a method for preparingR(+)-N-propargyl-1-aminoindan which comprises contacting, in thepresence of an organic or inorganic base, R(−)-aminoindan with eitherpropargyl bromide or propargyl chloride so as to formR(+)-N-propargyl-1-aminoindan, and isolating theR(+)-N-propargyl-1-aminoindan formed thereby.

[0054] The subject invention further provides a method for preparingracemic N-propargyl-1-aminoindan which comprises contacting, in thepresence of an organic or inorganic base, racemic 1-aminoindan withpropargyl bromide or propargyl chloride so as to form racemicN-propargyl-1-aminoindan, and isolating the racemicN-propargyl-1-aminoindan formed thereby.

[0055] Finally, the subject invention provides a method of preparing anR(+)-N-propargyl-1-aminoindan salt which comprises contacting racemicN-propargyl-1-aminoindan with an optically active acid so as to form twodiastereomeric N-propargyl-1-aminoindan salts, and isolatingR(+)-N-propargyl-1-aminoindan salt from the diastereomericN-propargyl-1-aminoindan salts so formed.

BRIEF DESCRIPTION OF THE FIGURES

[0056]FIG. 1 is a graphic representation of the results according toExample 22 showing in vitro MAO-A inhibitory activity.

[0057]FIG. 2 is a graphic representation of the results according toExample 22 showing in vitro MAO-B inhibitory activity.

[0058]FIG. 3 is a graphic representation of the results according toExample 22 showing MAO activity in human cortical tissue.

[0059]FIG. 4 is a graphic representation of the results according toExample 23 showing acute inhibition (i.p.) of MAO-A in brain.

[0060]FIG. 5 is a graphic representation of the results according toExample 23 showing acute inhibition (i.p.) of MAO-B in brain.

[0061]FIG. 6 is a graphic representation of the results according toExample 23 showing acute inhibition (i.p.) of MAO-A in liver.

[0062]FIG. 7 is a graphic representation of the results according toExample 23 showing acute inhibition (i.p.) of MAO-B in liver.

[0063]FIG. 8 is a graphic representation of the results according toExample 23 showing acute inhibition (per os) of MAO-A in brain.

[0064]FIG. 9 is a graphic representation of the results according toExample 23 showing acute inhibition (per os) of MAO-B in brain.

[0065]FIG. 10 is a graphic representation of the results according toExample 23 showing acute inhibition (per os) of MAO-A in liver.

[0066]FIG. 11 is a graphic representation of the results according toExample 23 showing acute inhibition (per os) of MAO-B in liver.

[0067]FIG. 2 is a graphic representation of the results according toExample 24 showing chronic inhibition of MAO-A in brain.

[0068]FIG. 13 is a graphic representation of the results according toExample 24 showing chronic inhibition of MAO-B in brain.

[0069]FIG. 14 is a graphic representation of the results according toExample 24 showing chronic inhibition of MAO-A in liver.

[0070]FIG. 15 is a graphic representation of the results according toExample 24 showing chronic inhibition of MAO-B in liver.

[0071]FIG. 16 is a graphic representation of the results according toExample 25 showing MAO-B activity in rat brain as a function of timefollowing i.p. administration of R(+)PAI.

[0072]FIG. 17 is a graphic representation of the results according toExample 32 showing restoration of normokinesia in mice that had receivedhaloperidol 6 mg/kg s.c. Mice received each of the test drugs i.p. atthe indicated dose. 2 hours later they received haloperidol. Kineticscores were taken 3 hours after haloperidol. These scores consisted ofthe ability to move horizontally along a rod, the ability to descend avertical rod, and the shortening of catalepsia. In the absence ofhaloperidol, the maximum score is 12, with haloperidol alone, 6.6±0.03.Statistical significance was calculated by the Student's “t” test:*p≦0.05; **p≦0.01; ***p≦0.001 with respect to haloperidol alone. Thescores of (R)-PAI are significantly different from those of racemic-PAIat 5 mg/kg (p≦0.05), at 10 mg/kg (p≦0.01), and at 15 mg/kg (p≦0.05),(n=5.6). The dosage shown is for the free base of PAI (and not themesylate salt).

[0073]FIG. 18 is a graphic representation of the results according toExample 32 showing restoration of motor activity in rats treated withα-methyl-p-tyrosine at 100 mg/kg i.p. Rats received the test drug i.p.at the indicated doses. After two hours they received α-Mpt and wereimmediately placed in activity cages. Total motor activity was recordedfor the duration of 10 hours. Control rats, treated with saline, onlyscored 15,862+1424. With α-Mpt alone, they scored 8,108±810. Statisticalsignificance by the Student's “t” test: *p≦0.05; **p≦0.01; ***p≦0.001with respect to α-MpT alone. The scores of (R)-PAI are significantlydifferent from racemic-PAI at 2 mg/kg (p≦0.01), (n=6). Dosage shown isfor the free base of PAI and not the mesylate salt.

[0074]FIG. 19 is a graph showing the NADH response to 2 minutes ofanoxia measured 30 minutes after injury and at half-hour intervalsthereafter.

[0075]FIG. 20: Ischemic brain lesion evaluation with MRI T2-scan 48hours after MCA-O and [R](+)PAI Mesylate Treatment in rats: The middlecerebral artery was surgically occluded as described in Example 38.[R](+)PAI Mesylate was administered as follows: 1.0 mg/kg ip immediatelyafter surgery; 0.5 mg/kg ip, 2 hrs after surgery; 1.0 mg/kg ip, 24 hrsafter surgery. Infarct volume (mm³) was determined by MRI 48 hoursfollowing surgery.

[0076]FIG. 21: Neurological evaluation of Wistar rats subjected to MCA-Oand [R](+)PAI Mesylate Treatment: The middle cerebral artery wassurgically occluded and [R](+)PAI Mesylate administered as in FIG. 20.At 24 hours post surgery a neurological score was taken as described inExample 38.

DETAILED DESCRIPTION OF THE INVENTION

[0077] The subject invention provides R(+)-N-propargyl-1-aminoindanhaving the structure:

[0078] As demonstrated in the Experimental Examples hereinbelow, R(+)PAIis nearly 7,000 times more active as an inhibitor of MAO-B than isS(−)PAI. In view of known MAO-B inhibitors in the art which possess lowselectivity between MAO-A and MAO-B, and which do not show predictabletrends in potency as a function of R or S configuration, the selectivityof R(+)PAI is unexpected.

[0079] R(+)PAI may be obtained by optical resolution of racemic mixturesof R- and S-enantiomers of PAI. Such a resolution can be accomplished byany conventional resolution method well known to a person skilled in theart, such as those described in J. Jacques, A. Collet and S. Wilen,“Enantiomers, Racemates and Resolutions,” Wiley, New York (1981). Forexample, the resolution may be carried out by preparative chromatographyon a chiral column. Another example of a suitable resolution method isthe formation of diastereomeric salts with a chiral acid such astartaric, malic, mandelic acid or N-acetyl derivatives of amino acids,such as N-acetyl leucine, followed by recrystallisation to isolate thediastereomeric salt of the desired R enantiomer.

[0080] The racemic mixture of R and S enantiomers of PAI may beprepared, for example, as described in GB 1,003,676 and GB 1,037,014.The racemic mixture of PAI can also be prepared by reacting1-chloroindan with propargylamine. Alternatively, this racemate may beprepared by reacting propargylamine with 1-indanone to form thecorresponding imine, followed by reduction of the carbon-nitrogen doublebond of the imine with a suitable agent, such as sodium borohydride.

[0081] In accordance with this invention, the R enantiomer of PAI canalso be prepared directly from the optically active R-enantiomer of1-aminoindan by reaction with propargyl bromide or propargyl chloride inthe presence of an organic or inorganic base, and optionally in thepresence of a suitable solvent.

[0082] Suitable organic or inorganic bases for use in the above reactioninclude, by way of example, triethylamine, pyridine, alkali metalcarbonates, and bicarbonates. If the reaction is conducted in thepresence of a solvent, the solvent may be chosen from, e.g., toluene,methylene chloride, and acetonitrile. One method of preparing R(+)PAI isto react R-1-aminoindan with propargyl chloride using potassiumbicarbonate as a base and acetonitrile as solvent.

[0083] The above-described reaction of 1-aminoindan generally results ina mixture of unreacted primary amine, the desired secondary amine andthe tertiary amine N,N-bispropargylamino product. The desired secondaryamine, i.e., N-propargyl-1-aminoindan, can be separated from thismixture by a conventional separation method including, by way ofexample, chromatography, distillation and selective extraction.

[0084] The R-1-aminoindan starting material can be prepared by methodsknown in the art which include, by way of example, the method of Lawsonand Rao, Biochemistry, 19, 2133 (1980), methods in references citedtherein, and the method of European Patent No. 235,590.

[0085] R-1-aminoindan can also be prepared by resolution of a racemicmixture of the R and S enantiomers, which involves, for example, thetormation of diastereomeric salts with chiral acids, or any other knownmethod such as those reported in J. Jacques, et al., ibid.Alternatively, R-1-aminoindan may be prepared by reacting 1-indanonewith an optically active amine, followed by reduction of the carbonnitrogen double bond of the resulting imine by hydrogenation over asuitable catalyst, such as palladium on carbon, platinum oxide or Raneynickel. Suitable optically active amines include, for example, one ofthe antipodes of phenethylamine or an ester of an amino acid, such asvaline or phenylalanine. The benzylic N—C bond may be cleavedsubsequently by hydrogenation under non-vigorous conditions.

[0086] An additional method for preparing R-1-aminoindan is thehydrogenation of indan-1-one oxime ethers as described above, whereinthe alkyl portion of the ether contains an optically pure chiral center.Alternatively, a non-chiral derivative of indan-1-one containing acarbon-nitrogen double bond, such as an imine or oxime, can be reducedwith a chiral reducing agent, e.g., a complex of lithiumaluminum-hydride and ephedrine.

[0087] The subject invention further provides a pharmaceuticallyacceptable salt of R(+)-N-propargyl-1-aminoindan.

[0088] In the practice of this invention, pharmaceutically acceptablesalts include, but are not limited to, the mesylate, maleate, fumarate,tartrate, hydrochloride, hydrobromide, esylate, p-toluenesulfonate,benzoate, acetate, phosphate and sulfate salts.

[0089] In one embodiment, the salt is selected from the group consistingof the mesylate salt of R(+)-N-propargyl-1-aminoindan, the esylate saltof R(+)-N-propargyl-1-aminoindan, and the sulfate salt ofR(+)-N-propargyl-1-aminoindan.

[0090] As demonstrated in the Experimental Examples hereinbelow, themesylate salt is highly stable to thermal degradation, and showsunexpectedly superior selectivity for MAO-B over the racemic salt.

[0091] For the preparation of pharmaceutically acceptable acid additionsalts of the compound of R(+)PAI, the free base can be reacted with thedesired acids in the presence of a suitable solvent by conventionalmethods. Similarly, an acid addition salt may be converted to the freebase form in a known manner.

[0092] A preferred mode of preparing the mesylate salt of (R)-PAIcomprises (a) adding an aqueous solution of 15% sodium hydroxide to asolution of propargyl benzenesulfonate (or tosylate or mesylate) intoluene; (b) stirring for 5 hours; (c) adding additional toluene andwater; (d) separating and washing the organic phase with 10% sodiumhydroxide, and then diluting with water; (e) adjusting the pH of themixture to 3.2 by adding 10% aqueous sulfuric acid; (f) separating theaqueous phase and adjusting the pH to 7.3 with 10% sodium hydroxide; (g)extracting three times with toluene while maintaining constant pH; (h)concentrating combined organic layers in vacuo to give a yellow oil; (i)dissolving the oil and L-tartaric acid in isopropanol; (j) heating toreflux for 1 hour; (k) cooling to room temperature and collecting theprecipitate by filtration; (l) recrystallizing the crudedi-propargylaminoindan tartrate from methanol/isopropanol (1:1) to givedi(R(+)-N-propargyl-1-aminoindan) tartrate; (m) dissolving the tartratesalt and methanesulfonic acid in isopropanol, and heating to reflux for30 minutes; and (n) cooling to room temperature, and collecting theprecipitated R(+)-N-propargyl-1-aminoindan.

[0093] The subject invention further provides a pharmaceuticalcomposition which comprises a therapeutically effective amount ofR(+)-N-propargyl-1-aminoindan or a pharmaceutically acceptable saltthereof and a pharmaceutically acceptable carrier. The “therapeuticallyeffective amount” of the R(+)-N-propargyl-1-aminoindan orpharmaceutically acceptable salt thereof may be determined according tomethods well known to those skilled in the art.

[0094] Possible salts useful for such compositions includehydrochloride, phosphate, maleate, fumarate, tartrate, mesylate,esylate, and sulfate salts.

[0095] These compositions may be prepared as medicaments to beadministered orally, parenterally, rectally, or transdermally.

[0096] In one embodiment, the pharmaceutically acceptable carrier is asolid and the pharmaceutical composition is a tablet. Thetherapeutically effective amount may be an amount from about 0.1 mg toabout 100 mg. The therapeutically effective amount may also be an amountfrom about 1 mg to about 10 mg.

[0097] Suitable forms for oral administration include tablets,compressed or coated pills, dragees, sachets, hard or soft gelatincapsules, sublingual tablets, syrups and suspensions.

[0098] In an alternative embodiment, the pharmaceutically acceptablecarrier is a liquid and the pharmaceutical composition is an injectablesolution. The therapeutically effective amount may be an amount fromabout 0.1 mg/ml to about 100 mg/ml. The therapeutically effective amountmay also be an amount from about 1 mg/ml to about 10 mg/ml. In oneembodiment, the dose administered is an amount between 0.5 ml and 1.0ml.

[0099] In a further alternative embodiment, the carrier is a gel and thepharmaceutical composition is a suppository.

[0100] For parenteral administration the invention provides ampoules orvials that include an aqueous or non-aqueous solution or emulsion. Forrectal administration there are provided suppositories with hydrophilicor hydrophobic vehicles. For topical application as ointments andtransdermal delivery there are provided suitable delivery systems asknown in the art.

[0101] In the preferred embodiment, the pharmaceutically acceptable saltis a mesylate salt.

[0102] These compositions may be used alone to treat the above-listeddisorders, or alternatively, as in the case of Parkinson's disease, forexample, they may be used as an adjunct to the conventional L-DOPAtreatments.

[0103] The preferred dosages of the active ingredient, i.e., R-PAI, inthe above compositions are within the following ranges. For oral orsuppository formulations, 0.1-100 mg per dosage unit may be taken daily,and preferably 1-10 mg per dosage unit is taken daily. For injectableformulations, 0.1-100 mg/ml per dosage unit may be taken daily, andpreferably 1-10 mg/ml per dosage unit is taken daily.

[0104] In one embodiment, the pharmaceutical composition furthercomprises a therapeutically effective amount of Levodopa. In anotherembodiment, the pharmaceutical composition still further comprises aneffective amount of a decarboxylase inhibitor.

[0105] The amount of decarboxylase inhibitor administered in combinationwith (R)-PAI or a pharmaceutically acceptable salt thereof is an amounteffective to ensure L-DOPA uptake in the subject.

[0106] The decarboxylase inhibitor may be L-Carbidopa. In oneembodiment, the therapeutically effective amount ofR(+)-N-propargyl-1-aminoindan is about 0.1 mg to about 100 mg, thetherapeutically effective amount of Levodopa is about 50 mg to about 250mg, and the effective amount of L-Carbidopa is about 10 mg to about 25mg.

[0107] The decarboxylase inhibitor may also be benserazide. In oneembodiment, the therapeutically effective amount ofR(+)-N-propargyl-1-aminoindan is about 0.1 mg to about 100 mg, thetherapeutically effective amount of Levodopa is about 50 mg to about 200mg, and the effective amount of benserazide is about 12.5 mg to about 50mg.

[0108] The subject invention further provides a method of treating asubject afflicted with Parkinson's disease which comprises administeringto the subject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat Parkinson's disease in the subject.

[0109] Methods of treatment of Parkinson's disease which combine the useof (R)-PAI with other drugs, such as dopamine agonists, bromocryptine,pergolide, lisuride, as well as catecholamine oxidase methyl transferaseinhibitors are within the scope of the subject invention.

[0110] In the preferred embodiment, the pharmaceutically acceptable saltis a mesylate salt.

[0111] The administering may comprise orally administering, rectallyadministering, transdermally administering, or parenterallyadministering.

[0112] In one embodiment, the method of the subject invention furthercomprises administering to the subject a therapeutically effectiveamount of Levodopa. In another embodiment, the method of the subjectinvention still further comprises administering to the subject aneffective amount of a decarboxylase inhibitor.

[0113] The decarboxylase inhibitor may be L-Carbidopa. Alternatively,the decarboxylase inhibitor may be benserazide.

[0114] The subject invention further provides a method of treating asubject afflicted with a memory disorder which comprises administeringto the subject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat the memory disorder in the subject.

[0115] The subject invention further provides a method of treating asubject afflicted with dementia which comprises administering to thesubject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat dementia in the subject. In one embodiment, thedementia is of the Alzheimer type (DAT).

[0116] The subject invention further provides a method of treating asubject afflicted with depression which comprises administering to thesubject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat depression in the subject.

[0117] The subject invention further provides a method of treating asubject afflicted with hyperactive syndrome which comprisesadministering to the subject an amount of R(+)-N-propargyl-1-aminoindanor the pharmaceutically acceptable salt thereof of the subject inventioneffective to treat hyperactive syndrome in the subject.

[0118] The administering may comprise orally administering, rectallyadministering, or parenterally administering.

[0119] The subject invention further provides a method of treating asubject afflicted with an affective illness which comprisesadministering to the subject an amount of R(+)-N-propargyl-1-aminoindanor the pharmaceutically acceptable salt thereof of the subject inventioneffective to treat the affective illness in the subject.

[0120] The subject invention further provides a method of treating asubject afflicted with a neurodegenerative disease which comprisesadministering to the subject an amount of R(+)-N-propargyl-1-aminoindanor the pharmaceutically acceptable salt thereof of the subject inventioneffective to treat tile neurodegenerative disease in the subject.

[0121] The subject invention further provides a method of treating asubject afflicted with a neurotoxic injury which comprises administeringto the subject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat the neurotoxic injury in the subject.

[0122] The subject invention further provides a method of treating asubject afflicted with brain ischemia which comprises administering tothe subject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat brain ischemia in the subject.

[0123] This invention provides a method of treating brain ischemia orstroke in a subject which comprises administering to the subject anamount of R(+)-N-propargyl-1-aminoindan or a pharmaceutically acceptablesalt thereof effective to treat brain ischemia or stroke in the subject.

[0124] In an embodiment of the method for treatment of brain ischemia orstroke, the pharmaceutically-acceptable salt ofR(+)-N-propargyl-1-aminoindan is selected from the group consisting of:the mesylate salt; the ethylsulfonate salt; the sulfate salt; and thehydrochloride salt. Preferably, the pharmaceutically acceptable salt isthe mesylate salt of R(+)-N-propargyl-1-aminoindan.

[0125] The effective amount can be determined using techniques known tothose of skill in the art, such as titration. In an embodiment of thisinvention, the effective amount is from about 0.5 milligrams perkilogram body weight of the subject to about 2.5 milligrams per kilogrambody weight of the subject. The R(+)-N-propargyl-1-aminoindan orpharmaceutically acceptable salt thereof is administered usingtechniques known to those of skill in the art. For example, it may beadministered intravenously, orally, rectally, transdermally, orparenterally.

[0126] The subject is preferably a mammal, such as a dog, cat, mouse,rat, rabbit, pig, horse, goat, sheep, cow, ape or monkey. In aparticular embodiment the subject is human.

[0127] In an embodiment of this invention, the effective amount is fromabout 0.01 mg to 50.0 mg per day. In a more specific embodiment, theeffective amount is from 0.1 to 10.0 mg per day.

[0128] In one embodiment of the above-described method, the area of isthe brain ischemia is reduced by about thirty-five percent.

[0129] The subject invention further provides a method of treating asubject afflicted with a head trauma injury which comprisesadministering to the subject an amount of R(+)-N-propargyl-1-aminoindanor the pharmaceutically acceptable salt thereof of the subject inventioneffective to treat the head trauma injury in the subject.

[0130] The subject invention further provides a method of treating asubject afflicted with a spinal trauma injury which comprisesadministering to the subject an amount of R(+)-N-propargyl-1-aminoindanor the pharmaceutically acceptable salt thereof of the subject inventioneffective to treat the spinal trauma injury in the subject.

[0131] This invention further provides a method of treating neurotraumain a subject which comprises administering to the subject an amount ofR(+)-N-propargyl-1-aminoindan or a pharmaceutically acceptable saltthereof effective to treat neurotrauma in the subject.

[0132] In the treatment of head trauma injury, spinal trauma injury orneurotrauma, the pharmaceutically acceptable salt ofR(+)-N-propargyl-1-aminoindan is selected from the group consisting of:the mesylate salt; the ethylsulfonate salt; the sulfate salt; and thehydrochloride salt. Preferably, the pharmaceutically acceptable salt isthe mesylate salt of R(+)-N-propargyl-1-aminoindan.

[0133] The effective amount can be determined using techniques known tothose of skill in the art, such as titration. In an embodiment of thisinvention, the effective amount is from about 0.5 milligrams perkilogram body weight of the subject to about 2.5 milligrams per kilogrambody weight of the subject. The R(+)-N-propargyl-1-aminoindan orpharmaceutically acceptable salt thereof is administered usingtechniques known to those of skill in the art. For example, it may beadministered intravenously, orally, rectally, transdermally, orparenterally.

[0134] The subject is preferably a mammal, such as a dog, cat, mouse,rat, rabbit, pig, horse, goat, sheep, cow, ape or monkey. In aparticular embodiment the subject is human.

[0135] In an embodiment of this invention, the effective amount is fromabout 0.01 mg to 50.0 mg per day. In a more specific embodiment, theeffective amount is from 0.1 to 10.0 mg per day.

[0136] The subject invention further provides a method of treating asubject afflicted with schizophrenia which comprises administering tothe subject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat schizophrenia in the subject.

[0137] The subject invention further provides a method of treating asubject afflicted with an attention deficit disorder which comprisesadministering to the subject an amount of R(+)-N-propargyl-1-aminoindanor the pharmaceutically acceptable salt thereof of the subject inventioneffective to treat the attention deficit disorder in the subject.

[0138] The subject invention further provides a method of treating asubject afflicted with multiple sclerosis which comprises administeringto the subject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat multiple sclerosis in the subject.

[0139] The subject invention further provides a method of preventingnerve damage in a subject which comprises administering to the subjectan amount of R(+)-N-propargyl-1-aminoindan or the pharmaceuticallyacceptable salt thereof of the subject invention effective to preventnerve damage in the subject.

[0140] In one embodiment, the nerve damage is structural nerve damage.In another embodiment, the structural nerve damage is optic nervedamage.

[0141] The subject invention further provides a method of treating asubject suffering from symptoms of withdrawal from an addictivesubstance which comprises administering to the subject an amount ofR(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable saltthereof of the subject invention effective to treat the symptoms ofwithdrawal in the subject.

[0142] As used herein, the term “symptoms of withdrawal” refers tophysical and/or psychological symptoms, including drug craving,depression, irritability, anergia, amotivation, appetite change, nausea,shaking and sleep irregularity.

[0143] As used herein, the term “addictive substance” includes, by wayof example, (a) addictive opiates such as opium, heroin and morphine,(b) psychostimulants such as cocaine, amphetamines and methamphetamines,(c) alcohol, (d) nicotine, (e) barbiturates and (f) narcotics such asfentanyl, codeine, diphenoxylate and thebaine.

[0144] In one embodiment, the addictive substance is cocaine. In anotherembodiment, the addictive substance is alcohol.

[0145] The subject invention further provides a method for preparingR(+)-N-propargyl-1-aminoindan which comprises contacting, in thepresence of an organic or inorganic base, R(−)-aminoindan with eitherpropargyl bromide or propargyl chloride so as to formR(+)-N-propargyl-1-aminoindan, and isolating theR(+)-N-propargyl-1-aminoindan formed thereby.

[0146] The subject invention further provides a method for preparingracemic N-propargyl-1-aminoindan which comprises contacting, in thepresence of an organic or inorganic base, racemic 1-aminoindan withpropargyl bromide or propargyl chloride so as to form racemicN-propargyl-1-aminoindan, and isolating the racemicN-propargyl-1-aminoindan formed thereby.

[0147] Finally, the subject invention provides a method of preparing anR(+)-N-propargyl-1-aminoindan salt which comprises contacting racemicN-propargyl-1-aminoindan with an optically active acid so as to form twodiastereomeric N-propargyl-1-aminoindan salts, and isolatingR(+)-N-propargyl-1-aminoindan salt from the diastereomericN-propargyl-1-aminoindan salts so formed.

[0148] In one embodiment, the isolating comprises isolating byfractional crystallization.

[0149] The following Experimental Details are set forth to aid in anunderstanding of the invention, and are not intended, and should not beconstrued, to limit in any way the invention set forth in the claimswhich follow thereafter.

EXPERIMENTAL DETAILS EXAMPLE 1 Racemic N-propargyl-1-aminoindanhydrochloride

[0150] 10.0 g of racemic 1-aminoindan and 10.4 g of potassium carbonatewere added to 75 ml of acetonitrile. The resulting suspension was heatedto 60° C. and 4.5 g of propargyl chloride was added dropwise.

[0151] The mixture was stirred at 60° C. for 16 hours, whereafter mostof the volatiles were removed by distillation in vacuo. The residue waspartitioned between 10% aqueous sodium hydroxide and methylene chloride.

[0152] The organic phase was dried and the solvent removed bydistillation. The residue was flash chromatographed on silica gel,eluting with 40% ethyl acetate/60% hexane. The fractions containing thetitle compound as a free base were combined and the eluant replaced byether. The ethereal solution was treated with gaseous HCl, theprecipitate formed was isolated by suction filtration and recrystallizedfrom isopropanol to yield 7.3 g of the title compound, m.p. 182-4° C.

[0153] Chromatographic and spectroscopic data were in accordance withU.S. Pat. No. 3,513,244, issued May 19, 1970, and an authentic sample,and were as follows: NMR δ(CDCl₃): 2.45 (2H, m), 2.60 (1H, t) , 2.90(1H, m), 3.45 (1H, m), 3.70 (2H, d), 4.95 (1H, t), 7.5 (4H, m) ppm.

EXAMPLE 2 S-(−)-N-Propargyl-1-aminoindan hydrochloride

[0154] The title compound in free base form was isolated by resolvingthe racemic mixture of the free base of Example 1 on a Chiracel OJ(cellulose tris [p-methylbenzoate]) preparative HPLC column eluting with10% isopropanol/90% hexane and collecting the first eluted major peak.The resulting oil was converted to the title compound (hydrochloride) bytreatment of a 10% diethyl ether solution of the oil with gaseous HCl,and the resulting precipitate was collected by suction filtration.[a]_(D) −29.2° (1%, ethanol), m.p. 182-184° C. Other chromatographic andspectroscopic properties were identical with the hydrochloride salt ofExample 1.

EXAMPLE 3 R-(+)-N-Propargyl-1-aminoindan hydrochloride

[0155] The title compound was prepared as in Example 2 above, exceptthat the second eluted peak from the preparative HPLC was collected:[a]_(D)+29.1° (0.8%, ethanol), m.p. 179-181° C. Other chromatographicand spectroscopic properties were identical with the hydrochloride saltof Example 1.

EXAMPLE 4 R-(+)-N-Propargyl-1-aminoindan hydrochloride

[0156] 12.4 g of R-(−)-1-Aminoindan and 12.9 g of potassium carbonatewere added to 95 ml of acetonitrile. The resulting suspension was heatedto 60° C. and 5.6 g of propargyl chloride was added dropwise. Themixture was stirred at 60° C. for 16 hours, whereafter most of thevolatiles were removed by distillation in vacuo. the residue waspartitioned between 10% aqueous sodium hydroxide and methylene chloride.

[0157] The organic phase was dried and the solvent removed in vacuo. Theresidue was flash chromatographed on silica get eluting with 40% ethylacetate/60% hexane. Fractions containing the free base of the titlecompound were combined and the solvent replaced by ether. The etherealsolution was treated with gaseous HCl and the resulting precipitate wasisolated by suction filtration and recrystallized from isopropanol toyield 6.8 g of the title compound, m.p. 183-185° C., [a]_(D)+30.90 (2%ethanol). Spectral properties were identical to those reported for thecompound of Example 1.

EXAMPLE 5 S-(−)-N-Propargyl-1-aminoindan hydrochloride

[0158] The title compound was prepared by the method of Example 4,except that S-(+)-1-aminoindan was used as starting material. Theproduct exhibited [a]_(D)−30.3 (2% ethanol), m.p. 183-5° C. Spectralproperties were identical to those reported for the compound of Example1.

EXAMPLE 6A Di(R-(+)-N-propargyl-1-aminoindan) L-tartrate

[0159] To a solution of tartaric acid (4.4 g) in 48 ml of boilingmethanol was added a solution of R-(+)-N-propargyl-1-aminoindan freebase (5.0 g) in methanol (48 ml). The solution was heated to reflux and284 ml of t-butylmethyl ether was added over 20 minutes. The mixture washeated for an additional 30 minutes, cooled, and the resultingprecipitate was isolated by suction filtration to yield 6.7 g of thetitle compound: m.p. 175-177° C.; [α]_(D) (1.5, H₂O)=+34.3; Anal. calcd.for C₂₈H₃₂O₆N₂; C, 68.26, H, 6.56, N, 5.69. Found: C, 68.76; H, 6.57; N,5.61.

EXAMPLE 6B R-(+)-N-propargyl-1-aminoindan mesylate

[0160] a) To a solution of propargyl benzenesulfonate (78.4 g) andracemic aminoindan (63.2 g) in toluene (240 mL) at 20° C. was addeddropwise an aqueous solution of 15% sodium hydroxide (108 mL). After 5hours of stirring, additional toluene (80 mL) and water (200 mL) wereadded with stirring. The organic phase was separated and washed with 10%aqueous sodium hydroxide and then diluted with water. The pH of themixture was adjusted to 3.2 by the addition of 10% aqueous sulfuricacid. The aqueous phase was separated and its pH was adjusted to 7.3with 10% sodium hydroxide and extracted three times with toluene whilemaintaining constant pH. The combined organic layers were concentratedin vacuo to 40.7 g of a yellow oil.

[0161] b) The above crude racemic propargylaminoindan and L-tartaricacid (log) were dissolved in isopropanol (1 L) and heated to reflux for1 hour. The reaction was then allowed to cool to room temperature withstirring and the precipitate collected by filtration. The crudedi-propargylaminoindan tartrate was recrystallized from 1 L of 1;1methanol/isopropanol to givedi(R-(+)-N-propargyl-1-aminoindan)-L-tartrate with physical and spectralproperties identical to that of the compound of Example 6A.

[0162] c) A solution of di-(R-(+)-N-propargyl-1-aminoindan) tartrate (15g) and methanesulfonic acid (6 g) in isopropanol (150 mL) was heated toreflux for 30 minutes. The reaction was allowed to cool to roottemperature and the resulting precipitate isolated by suction filtrationto give the title compound (11.1 g) with m.p. 157° C. and [α]_(D)=22°.

EXAMPLE 7 R-(+)-N-Methyl-N-propargyl-1-aminoindan hydrochloride

[0163] The free base form of R-(+)-N-propargyl-1-aminoindan from Example4 (1.2 grams), potassium carbonate (0.97 grams) and methyl iodide (1gram) were added to 15 ml of acetone and the resulting suspension heatedto reflux under a nitrogen atmosphere for 8 hours. Thereafter thevolatiles were removed under reduced pressure and the residuepartitioned between 10% aqueous sodium hydroxide (30 ml) and methylenechloride (30 ml). The organic phase was dried and the solvent removed invacuo. The residue was flash chromatographed on silica gel eluting with40% ethyl acetate/60% hexane. Fractions containing the title compound asa free base were combined and the solvent replaced by diethyl ether. Theetheral solution was treated with gaseous HCl. The volatiles wereremoved in vacuo, and the residue recrystallized from isopropanol toyield 400 mg of the title compound as a white crystalline solid, m.p.134-136° C., [α]_(D)+31.40 (ethanol). NMR δ(CDCl₃): 2.55 (2H, m); 2.7(1H, br.s); 2.8 (3H, s); 3.0 (1H, m); 3.4 (1H, m); 3.9 (2H, br.s); 5.05(1H, m); 7.7 (4H, m) ppm.

EXAMPLE 8 S-(−)-N-Methyl-N-prorargyl-1-aminoindan hydrochloride

[0164] The title compound was prepared as in Example 7 above, exceptthat S-(−)-N-propargyl-1-aminoindan (free base) from Example 5 was usedas the starting material. All of the physical and spectral properties ofthe title compound were identical to those in Example 7 except for the[α]_(D) −34.9° C. (ethanol). EXAMPLE 9 Tablet CompositionN-Propargyl-1(R)-aminoindan Hydrochloride 7.81 mg* Pregelatinized starchNF 47.0 mg Lactose NF hydrous 66.0 mg Microcrystalline cellulose NF 20.0mg Sodium starch glycolate NF 2.99 mg Talc USP 1.5 mg Magnesium stearateNF 0.7 mg *Equivalent to 5.0 mg of N-propargyl aminoindan base. EXAMPLE10 Tablet Composition N-Propargyl-1(R)-aminoindan Hydrochloride 1.56 mg*Lactose hydrous 50.0 mg Pregelatinized starch 36.0 mg Microcrystallinecellulose 14.0 mg Sodium starch glycolate 2.14 mg Talc USP 1.0 mgMagnesium stearate NF 0.5 mg *Equivalent to 1.0 mg of N-propargylaminoindan base. EXAMPLE 11 Capsule CompositionN-Propargyl-1(R)-aminoindan Hydrochloride 5.0 mg Pregelatinized starch10.0 mg Starch 44.0 mg Microcrystalline cellulose 25.0 mg Ethylcellulose1.0 mg Talc 1.5 mg Purified water added as required for granulation.EXAMPLE 12 Injection Composition N-Propargyl-1(R)-aminoindanHydrochloride 5.0 mg Dextrose anhydrous 44.0 mg HCl added to pH 5Purified water added as required for 1 ml EXAMPLE 13 InjectionComposition N-Propargyl-1(R)-aminoindan Hydrochloride 1.0 mg Sodiumchloride 8.9 mg HCl added to pH 5 Purified water added as required for 1ml EXAMPLE 14 Injection Composition N-Propargyl-1(R)-aminoindanHydrochloride 2.0 mg Sodium chloride 8.9 mg HCl added to pH 5 Purifiedwater added as required for 1 ml EXAMPLE 15 Syrup CompositionN-Propargyl-1(R)-aminoindan Hydrochloride 5.0 mg Sucrose 2250.0 mgSaccarin sodium 5.0 mg Methylparaben 6.0 mg Propylparaben 1.0 mg Flavor20.0 mg Glycerin USP 500 mg Alcohol 95% USP 200 mg Purified water asrequired to 5.0 ml EXAMPLE 16 Sublingual TabletsN-Propargyl-1(R)-aminoindan Hydrochloride 2.5 mg Microcrystallinecellulose 20.0 mg Lactose hydrous 5.0 mg Pregelatinized starch 3.0 mgPovidone 0.3 mg Coloring agent q.s. Flavor q.s. Sweetener q.s. Talc 0.3mg Blend the excipients and the active and granulate with an ethanolsolution of Providone. After drying and weighing, it is blended with thetalc and compressed. EXAMPLE 17 PAI Sublingual TabletsN-Propargyl-1(R)-aminoindan Hydrochloride 5.0 mg Microcrystallinecellulose 15.0 mg Pregelatinized starch 12.0 mg Ethyl cellulose 0.3 mgTalc 0.3 mg Purified water added as required for granulation. EXAMPLE 18Tablet Composition N-Propargyl-1(R)-aminoindan Hydrochloride 5.0 mgLevodopa 100.0 mg Carbidopa 25.0 mg Pregelatinized starch 24.0 mg Starch40.0 mg Microcrystalline cellulose 49.5 mg Col. D & C Yellow No. 10 0.5mg Col. D & C Yellow No. 6 0.02 mg Alcohol USP added as required forgranulation. EXAMPLE 19 Tablet Composition N-Propargyl-1(R)-aminoindanMesylate 7.81 mg* Pregelatinized starch NF 47.0 mg Lactose NF hydrous66.0 mg Microcrystalline cellulose NF 20.0 mg Sodium starch glycolate NF2.99 mg Talc USP 1.5 mg Magnesium stearate NF 0.7 mg *Equivalent to 5.0mg of N-propargyl aminoindan base. EXAMPLE 20 Tablet CompositionN-Propargyl-1(R)-aminoindan Mesylate 1.56 mg* Lactose hydrous 50.0 mgPregelatinized starch 36.0 mg Microcrystalline cellulose 14.0 mg Sodiumstarch glycolate 2.14 mg Talc USP 1.0 mg Magnesium stearate NF 0.5 mg*Equivalent to 1.0 mg of N-propargyl aminoindan base. EXAMPLE 21 CapsuleComposition N-Propargyl-1(R)-aminoindan Mesylate 5.0 mg Pregelatinizedstarch 10.0 mg Starch 44.0 mg Microcrystalline cellulose 25.0 mgEthylcellulose 1.0 mg Talc 1.5 mg Purified water added as required forgranulation.

[0165] The following Examples and the accompanying Tables and Figuresrelate to biological experiments carried out in accordance with thisinvention.

EXAMPLE 22 Inhibition of MAO Activity in vitro

[0166] Experimental Protocol

[0167] The MAO enzyme source was a homogenate of rat brain in 0.3Msucrose, which was centrifuged at 600 g for 15 minutes. The supernatantwas diluted appropriately in 0.05M phosphate buffer, and pre-incubatedwith serial dilutions of compounds: R(+)-PAI, S(−)-PAI and racemic PAIfor 20 minutes at 37° C. ¹⁴C-Labelled substrates (2-phenylethylamine,hereinafter PEA; 5-hydroxytryptamine, hereinafter 5-HT) were then added,and the incubation continued for a further 20 minutes (PEA), or 30-45minutes (5-HT). Substrate concentrations used were 50 uM (PEA) and 1 mM(5-HT). In the case of PEA, enzyme concentration was chosen so that notmore than 10% of the substrate was metabolized during the course of thereaction. The reaction was then stopped by addition of tranylcypromine(to a final concentration of 1 mM), and the incubate filtered over asmall column of Amberlite CG-50 buffered to pH 6.3. The column waswashed with 1.5 ml water, the eluates pooled and the radioactive contentdetermined by liquid scintillation spectrometry. Since the aminesubstrates are totally retained on the column, radioactivity in theeluate indicates the production of neutral and acidic metabolites formedas a result of MAO activity. Activity of MAO in the sample was expressedas a percentage of control activity in the absence of inhibitors aftersubtraction of appropriate blank values. The activity determined usingPEA as substrate is referred to as MAO-B, and that determined using 5-HTas MAO-A.

[0168] Results

[0169] Inhibitory activity of R(+)-PAI, S(−)-PAI and racemic-PAI wereexamined separately in vitro, and the results of typical experimentalruns are shown in FIGS. 1 and 2. The entire experiment was repeatedthree times. Concentrations of inhibitor producing 50% inhibition ofsubstrate metabolism (IC-50) were calculated from the inhibition curves,and are shown in Table 1B. From this data it can be seen that:

[0170] (a) the R(+)-PAI is twice as active as the racemate forinhibition of MAO-B;

[0171] (b) the R(+)-PAI is 29 times more active for inhibition of MAO-Bthan MAO-A;

[0172] (c) the S(−)-PAI is only 1/6,800 as active as the R(+)PAI forinhibition of MAO-B, and shows little or no selectivity between MAO-Band MAO-A. TABLE 1A IC-50 (nM) VALUES FOR INHIBITION OF MAO-A AND MAO-BBY RACEMIC-PAI AND THE R(+) AND S(−) ENANTIOMERS THEREOF IN RAT BRAINHOMOGENATE IN VITRO MAO-A IC-50 (nM) MAO-B S (−) PAI R (+) PAI Rac S (−)PAI R (+) PAI Rac 26000 73 140 17000 2.5 5

[0173] The results of the same experiments using R(+) and S(−) MPAI(N-methyl-N-propargyl-1-aminoindan) are reported in Table 1B. Each ofthe enantiomers of MPAI is less selective in MAO-A and MAO-B inhibitionthan R(+)PAI. Furthermore, R(+)-MPAI is only five times as active asS(−)-MPAI in MAO-B inhibition, in contrast to R(+)-PAI which is about7000 times as active as S(−)-PAI in this assay. TABLE 1B IC-50 (nM)VALUES FOR INHIBITION OF MAO-A AND MAO-B BY THE R(+) AND S(−)ENANTIOMERS OF MPAI IN RAT BRAIN HOMOGENATE IN VITRO IC-50 (nM) MAO-AMAO-B Compound: S(−) MPAI R(+) MPAI S(−) MPAI R(+) MPAI 70 3 50 10

[0174] Some experiments were also carried out with human cerebralcortical tissues obtained 6 hours post-mortem, and treated as describedabove. The results of such an experiment are shown in FIG. 3, whereR(+)-PAI, S(−)-PAI, and racemic PAI are as defined herein.

EXAMPLE 23 Inhibition of MAO Activity in vivo: Acute Treatment

[0175] Experimental Protocol

[0176] Rats (male Sprague-Dawley-derived) weighing 250±20 g were treatedwith one of the enantiomers or the racemic form of PAI byintraperitoneal injection (ip) or oral gavage (po) and decapitated 1 hor 2 h later respectively. Groups of three rats were used for each doselevel of inhibitor, and MAO activity determined in brain and liver usingthe general technique described above. The amount of protein in eachincubation was determined using the Folin-Lowry method, and enzymeactivity calculated as nmol of substrate metabolized per hour ofincubation for each mg of protein. Activity of MAO in tissues fromanimals treated with inhibitors was expressed as a percentage of theenzyme activity in a group of control animals administered vehicle(water for oral administration, 0.9% saline for ip injection) and killedas above.

[0177] Results

[0178] None of the dose levels used with the inhibitor drugs producedany obvious behavioral alteration. The results are depicted in FIGS. 4to 11. Following i.p. administration, compound R(+)PAI produced 90%inhibition of brain MAO-B activity at a dose of 0.5 mg/kg. The same doseproduced only 20% inhibition of MAO-A activity. By oral administration,the same dose of R(+)PAI produced 80% inhibition of MAO-B with nodetectable inhibition of MAO-A. Essentially similar results were seenfor inhibition of hepatic MAO, as for brain MAO. The doses producing 50%inhibition of MAO-A and MAO-B (IC-50) were calculated from theinhibition curves, and are shown in Table 2. These data show: (a) thatMAO inhibitory activity of R(+)PAI is maintained in vivo in the rat; (b)that selectivity for inhibition of MAO-B, as opposed to MAO-A, byR(+)PAI is maintained in vivo; (c) that the much greater activity of the(+)-enantiomer as opposed to the (−)-enantiomer, is maintained in vivo;(d) that the compounds are effectively absorbed after oraladministration; and (e) that the compounds effectively pass theblood-brain barrier, and effectively inhibit brain MAO. The fact thatR(+)-PAI was about twice as active as the racemic compound forinhibition of MAO-B is a reflection of the extremely low activity ofS(−)-PAI for inhibition of MAO-B. TABLE 2 IC-50 VALUES (mg/kg) FORINHIBITION OF MAO-A AND MAO-B BY R(+)-PAI, S(−)-PAI OR RACEMIC-PAI, INTHE RAT FOLLOWING INTRAPERITONEAL (I.P.) INJECTION OR ORALADMINISTRATION (P.O.) IC-50 (mg/kg) MAO-A S (−) MAO-B Compound: PAI R(+) PAI Rac S (−) PAI R (+) PAI Rac I.P. BRAIN >10 1.2 2.5 >10 0.07 0.22I.P. LIVER >10 5 5 >10 0.06 0.11 P.O. BRAIN >10 >5 >5 >10 0.17 0.29 P.O.LIVER >10 >5 >5 >10 0.05 0.09

EXAMPLE 24 Inhibition of MAO Activity in vivo: Chronic Treatment

[0179] Experimental Protocol

[0180] Rats (specifications as in Example 23, 4 animals for each doselevel) were treated with R(+)PAI or the racemic mixture at three doselevels (0.05, 0.1 and 0.5 mg/kg) by oral administration, one dose dailyfor 21 days, and decapitated 2 hours after the last dose. The activitiesof MAO types A and B were determined in brain and liver as described inExample 23.

[0181] Results

[0182] A daily dose of 0.1 mg/kg of compound R(+)PAI produced a gooddegree of selective inhibition, with more than 80% inhibition of brainMAO-B and 20% or less inhibition of brain MAO-A. At the higher dose of0.5 mg/kg daily, MAO-A was still inhibited by less than 50% (FIGS. 12and 13). Hepatic MAO showed a similar degree of selective inhibition(FIGS. 14 and 15). Compound R(+)PAI was again more potent than theracemic mixture by a factor of about twofold. In the case of brain MAO,R(+)PAI had a better degree of selectivity for inhibition of MAO-B thandid the racemic mixture.

[0183] These results show that selectivity of MAO-B inhibition can bemaintained following chronic treatment with the compounds. As with otherirreversible inhibitors, the degree of enzyme inhibition is greater withchronic treatments than that following a single dose of the drug.Compound R(+)PAI shows a better degree of selectivity for inhibition ofbrain MAO-B than the racemic mixture.

EXAMPLE 25 Irreversible Nature of MAO Inhibition

[0184] Experimental Protocol

[0185] A single dose of compound R(+)PAI (1 mg/kg) was administered byi.p. injection to groups of 4 rats, and the animals killed 2, 6, 18, 24,48 and 72 hours later. Activity of MAO-B was determined in whole braintissues as described hereinabove.

[0186] Results

[0187] The results are shown in FIG. 16. Maximal inhibition of MAO-B wasattained at 6 hours after injection. MAO activity had only returned to30% of control activity at 72 hours after injection. This experimentdemonstrates the irreversible nature of the MAO inhibition by R(+)PAI.

EXAMPLE 26 Potentiation of Tyramine Pressor Effect in Conscious Rats

[0188] Experimental Protocol

[0189] Rats were anesthetized with a mixture of pentobarbital (30 mg/kg)and chloral hydrate (120 mg/kg) by intraperitoneal injection. The leftcarotid artery and jugular vein were cannulated with fine polytenetubing (artery) or fine silicone rubber tubing connected to polyethylenetubing (vein), the distal end of which was brought under the skin to ananchor point behind the neck. The tubing was filled with heparinizedsaline solution, and plugged with a fine steel rod. The animals weretreated with 2.0 mg chloramphenicol by intramuscular injection andallowed to recover from the operation overnight. The following day, therats were placed in a high-walled container permitting free movement.The arterial catheter was connected to a pressure transducer via a 100cm length of saline-filled, fine-bore polyethylene tubing, and thevenous catheter connected to a 1 ml syringe via a similar length oftubing, which, together with the syringe, contained a solution oftyramine hydrochloride in saline (1 mg/ml). Following an equilibrationperiod of 30 to 40 minutes, tyramine injections (50 or 100 μg) weregiven, and blood pressure responses recorded. An interval of at least 15minutes was maintained between injections after return of blood pressureto control values. Control pressor responses were established, then oneof the drugs was injected intraperitoneally, and tyramine responses wererepeated over the next 4 hours. The area under the blood pressureresponse curve was estimated, and the ratio of this area after treatmentto before treatment and to 1 to 3 hours after injection of thecompounds, was determined using the average of 3 to 4 values obtained inthe control period.

[0190] Results

[0191] The results are shown in Table 3. Compound R(+)PAI at a dose of 1mg/kg (which causes complete inhibition of MAO-B in brain and liver, and40 to 50% inhibition of MAO-A in these tissues) caused no significantpotentiation of tyramine pressor response. At the higher R(+)PAI dose of5 mg/kg (which causes more extensive inhibition of MAO-A in brain andperiphery), there was a significant potentiation of the tyramine pressorresponse, which was similar in extent to that produced by the same doseof deprenyl, and less than that produced by clorgyline (at a dose whichinhibits hepatic MAO-A activity by over 85%). TABLE 3 POTENTIATION OFTYRAMINE PRESSOR EFFECT IN CONSCIOUS RATS BY MAO INHIBITORS Ratio AreaUnder Dose No. of rats Pressor Response Inhibitor (mg/kg) (n) Curve;After/Before SEM* Saline 12 1.25 0.28 Clorgyline 2 6 10.39 2.13 (−)Deprenyl 1 2 1.15 (+) Deprenyl 5 3 2.36 0.16 R (+) PAI 1 3 1.38 0.7 R(+) PAI 5 3 3.49 0.98

[0192] From this experiment it can be concluded that compound R(+)PAIcauses no potentiation of the tyramine pressor effect at a dose whicheffectively inhibits MAO-B.

EXAMPLE 27 Suppression of MPTP-induced Dopaminergic Toxicity by R(+)PAI

[0193] 1-Methyl-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxinthat damages nigrostriatal dopaminergic neurons in several mammalianspecies, including mice, and produces a Parkinsonian syndrome in humansand primates. A crucial initial step in the mechanism of itsneurotoxicity involves conversion of MPTP to its toxic metabolite1-methyl-4-phenyl pyridinium ion (MPP+). This reaction is catalyzed bythe enzyme MAO-B and probably takes place outside of dopaminergicneurons, mainly in glia. It is known that MPTP is both a substrate andan irreversible inhibitor of MAO-B. Pretreatment of experimental animalswith MAO-B inhibitors such as deprenyl or pargyline protects against andprevents the MPTP-induced damage to nigrostriatal neurons because theoxidative conversion of MPTP to MPP+ is blocked. The progressivenigrostriatal degeneration in Parkinson's may be due to exposure toenvironmentally-derived exogenous MPTP-like neurotoxins. In such cases,there is an additional strong indication of initiation of sustainedtreatment with an MAO-B inhibitor from the very early stages ofParkinson's disease in the hope that it will neutralize the damagingeffects of such yet putative MPTP-toxins, and thus arrest or slow downthe progression of the illness. A successful MAO-B inhibitor drug iscurrently judged by its ability to block MPTP-induced damage tonigrostriatal dopaminergic neurons in vivo. The (−) and (+) enantiomersof PAI were therefore tested for their potency in preventing orattenuating the MPTP-induced striatal dopamine depletions in mice.

[0194] Experimental Protocol

[0195] Male C57 black mice (20-25 g weight) were (a) injected withMPTP.HCl (30 mg/kg dissolved in distilled water, s.c.), or vehiclealone, or one hour after pretreatment with the (−) or (+) isomers of PAI(2.5 mg/kg, i.p.), or with deprenyl (5 mg/kg, i.p.), and (b) decapitated5 days later. Brains were removed and corpora striata dissected on anice-cold glass plate and frozen on dry ice. Striatal tissues werehomogenized in 0.1 M perchloric acid, and deproteinized aliquotscontaining dihydroxybenzylamine as an internal standard were assayed fordopamine and its major metabolite 3,4-dihydroxy-phenylacetic acid(DOPAC) using HPLC with electrochemical detection.

[0196] Results

[0197] Table 4 shows the results of this experiment. Treatment with MPTPalone produced marked striatal dopamine (DA) and DOPAC depletions.Treatment with the (−) and (+) enantiomers of PAI or with (−) deprenyldid not affect striatal DA concentrations. Pretreatment with the (−)isomer of PAI did not affect the MPTP-induced DA and DOPAC levels in thestriatum. The (+)-isomer of PAI given before MPTP completely abolishedthe reduction in striatal DA and DOPAC levels produced by the toxin. Ata dose of 2.5 mg/kg, (+)PAI was equipotent to (−) deprenyl (5 mg/kg) inits protective effect. TABLE 4 EFFECT OF PRETREATMENT WITH THE (−) AND(+) ENANTIOMERS OF THE MAO-B INHIBITOR PAI ON THE STRIATAL DA AND DOPACDEPLETIONS INDUCED BY MPTP IN MICE IN VIVO DA DOPAC (ng/mg protein)Control 162.8 ± 7.2  8.4 ± 0.5 MPTP 53.1 ± 6.2 3.2 ± 0.3 (−) PAI 174.0 ±4.8  7.5 ± 0.2 (−) PAI + MPTP 53.4 ± 6.9 7.0 ± 0.6 (+) PAI 185.0 ± 6.9 3.3 ± 0.3 (+) PAI + MPTP 177.8 ± 14.4 6.0 ± 0.3 (−) Deprenyl 170.6 ±7.1  5.6 ± 0.3 (−) Deprenyl + MPTP 197.0 ± 8.0  6.4 ± 0.5

[0198] Above values for DA and DOPAC expressed as Mean±S.E.M. and numberof rats. n=7-11 in each group.

[0199] These results indicate that the R(+)PAI is an excellent MAO-Binhibitor in vivo, and is of especially great potential for thetreatment of Parkinson's disease.

[0200] While the invention has been described with reference to theaforementioned Examples and the accompanying Tables and Figures, it isnot restricted thereto. Various modifications and applications of theinvention are possible. For example, (R)-PAI may be combined, in asynergistic way, with α-tocopherol (a vitamin E derivative) for thetreatment of Parkinson's disease.

EXAMPLE 28 Effect of PAI Enantiomers on Amphetamine Induced StereotypeBehavior in Senescent Rats

[0201] Amphetamine is known to induce stereotypic behavior (Sulser, F.,and Sanders-Bush, E., Ann. Rev. Pharmacol., 11, 209-230 (1971)) by themobilization of endogenous dopamine. Amphetamine is not metabolized byMAO-B. Inhibition of MAO-B by an effective inhibitor and administrationof amphetamine cause release of dopamine which will not undergodegradation by the inhibited MAO-B. Thus, an increase of synapticdopamine is expected after administration of amphetamine and effectiveMAO-B inhibitor leading to an increase in stereotypebehavior-potentiation of the amphetamine effect. The extent of thisbehavior is rated in accordance with the number of lateral headmovements over a period of 1 minute.

[0202] Experimental Protocol

[0203] The test compound was administered at a dose of 0.5 mg/kg/day indrinking water, 24 hours before the infliction of hypoxia (92%nitrogen+8% oxygen for 6 hours). Following that, amphetamine wasinjected s.c. at a dose of 0.5 mg/kg. 45 minutes later, lateral headmovements were counted.

[0204] Results

[0205] The results of these experiments are shown in Table 5. TABLE 5EFFECT OF PAI ISOMERS ON AMPHETAMINE-INDUCED STEREOTYPE BEHAVIOR INSENESCENT RATS (CONTROL AND HYPOXIA LESIONED) Stereotype Group TreatmentBehavior Rating Control (6) — 87 ± 10 Control (5) (+) PAI 126 ± 16*Control (4) (−) PAI 94 ± 18 Hypoxia lesioned (5) — 93 ± 12 Hypoxialesioned (6) (+) PAI 143 ± 6* 

[0206] The results in Table 5 indicate that (+)PAI caused significantpotentiation of the amphetamine-induced stereotype behavior in bothhypoxia-lesioned and control rats. (−)PAI was totally inactive in thisrespect. These behavioral in vivo results corroborate previousbiochemical findings that (+)PAI is an active inhibitor of MAO-B in thebrain while (−)PAI is inactive in this respect.

EXAMPLE 29 Effect on R(+)-PAI on the Improvement or Restoration ofMemory

[0207] Newborn rat pups subjected to a brief episode of anoxia and thenallowed to resume their growth in a normal way develop a long-lastingimpairment of memory (Speiser, et al., Behav. Brain Res., 30, 89-94(1988)). This memory impairment is expressed as an inferior performancein the passive avoidance test.

[0208] The-effect of R(+)-PAI and S(−)-PAI on the improvement orrestoration of memory was investigated in the passive avoidance test. Ifthe drug is effective, it increases the latency of response to enter adark compartment or chamber where an electroshock has been experiencedearlier by the rat being tested. The latency of the maximal response is300 seconds.

[0209] Experimental Protocol

[0210] Young rats were subjected to post-natal anoxia as described inExample 27. R(+)-PAI or S(−)-PAI were administered according to one ofthe following protocols.

[0211] Protocol A—Nursing mothers were given a dose of either isomer of1-1.5 mg/kg/day in drinking water until weaning at 21 days. Followingthat, the weaned offsprings were directly treated with the same dose for20 days. Treatment was terminated at 40 days and the test was performedat 60 days, that is 20 days after the last dose of the drug.

[0212] Protocol B—The dose was reduced to 0.5 mg/kg/day administered tothe nursing mother until weaning at 21 days, then directly to the youngrats to 60 days at which time the test was performed.

[0213] Passive Avoidance Test—The apparatus consisted of a lit chamberadjoining a dark chamber and a sliding door separating the two. Attraining, a rat was placed in the lit chamber for 30 seconds, and thenthe door was opened. The rat moved to the dark chamber with a latencythat was recorded. Upon entry of the rat into the dark compartment, thedoor was closed and a 0.3 mA foot-shock was delivered for 3 seconds.

[0214] Retention (memory) after 48 hours was determined by repeating thetest and recording the latency to step through from light to darkness toan arbitrary maximum of 300 seconds.

[0215] Results

[0216] The results of these experiments are shown in Table 6. TABLE 6EFFECT OF PAI ISOMERS ON PASSIVE AVOIDANCE RESPONSE IN YOUNG RATS(60-DAYS OLD) Before After Group Treatment Electroshock ElectroshockPROTOCOL A Control — 49 ± 13 201 ± 111 Control (+) PAI 49 ± 19 220 ± 100(+9%)* Control (−) PAI 48 ± 13 192 ± 116 Anoxia-lesioned — 45 ± 11 183 ±109 Anoxia-lesioned (+) PAI 49 ± 10 239 ± 99 (19%)* Anoxia-lesioned (−)PAI 55 ± 27 179 ± 123 PROTOCOL B Control — 53 ± 20 104 ± 101 Control (+)PAI 48 ± 11 128 ± 119 (+23%)* Anoxia-lesioned — 45 ± 8 119 ± 105Anoxia-lesioned (+) PAI 52 ± 12 137 ± 126 (+15%)* Anoxia-lesioned (−)PAI 48 ± 19 112 ± 112

[0217] The experimental results indicated that (+)PAI but not (−) PAI iseffective in improving the memory of anoxia-lesioned and control rats.Drugs active in this test are considered to be potentially useful fortreatment of various memory impairment disorders, dementia andespecially senile dementia of the Alzheimer's type.

EXAMPLE 30 Effect of R(+)-PAI on the Anoxia-induced Hyperactive Syndromein Juvenile Rate

[0218] Rats that had been exposed postnatally to anoxia and then left togrow under normal conditions show increased motor activity in the openfield at the age of 10-42 days (Hertshkowitz, et al., Dev. Brain Res.,7, 145-155 (1983)).

[0219] The effect of R(+)PAI and S(−)PAI on such hyperactive syndromewas investigated.

[0220] Experimental Protocol

[0221] Anoxia was performed on rat pups on the first post-natal day.They were placed in a glass chamber and exposed to 100% nitrogen for 25minutes. They were resuscitated by intermittent massage softly appliedto the chest and then returned to their respective mothers. Control ratsreceived the same treatment but with air instead of nitrogen.

[0222] The R(+)-PAI or S(−)-PAI (0.5 mg/kg/day) was administered to thenursing mothers in drinking water, thereby transferred to the sucklingsthrough milk.

[0223] Locomotion was measured in 6 fully computerized cages (28×28 cm)by recording the number of crossings over a given period of time.Crossings of grid infrared beams at 4-cm intervals initiates electricalimpulses which fed a counter. Recordings of motor activity were made atthe ages of 15 and 20 days, over a period of 15 minutes.

[0224] Results

[0225] The experimental results are given in Table 7. TABLE 7 EFFECT OFEACH OF THE TWO ENANTIOMERS ON THE ANOXIA-INDUCED HYPERACTIVE SYNDROME15-day old 20-day old Group Treatment rats rats Control — 414 ± 192 (11)808 ± 212 (12) Control (+) PAI 254 ± 149 (11) c 719 ± 110 (13) Anoxia- —482 ± 119 (7) 858 ± 96 (9) lesioned Anoxia- (+) PAI 276 ± 186 (15) a 737± 150 (16) c lesioned Anoxia- (−) PAI 334 ± 196 (5) 778 ± 232 (6)lesioned

[0226] These results indicate that chronic oral treatment with R(+)-PAIat a dose of 0.5 mg/kg administered to the nursing mother and reachingthe milk-fed offspring significantly improved the hyperactive syndrome.Consequently, R(+)-PAI is a potentially useful drug for the treatment ofthe hyperactive syndrome in children.

EXAMPLE 31 Stability Differences Among Ten Salts of PAI

[0227] Stability is an important factor in the selection of an optimalsalt as a therapeutic drug. Different salts may alter thephysicochemical and biological characteristics of a drug and can have adramatic influence on its overall properties. (Berge, S. M., et al., J.Pharm. Sci. 66, 1 (1977); Gould, P. L., Int. J. Pharmaceutics, 33, 201(1986)).

[0228] Experimental

[0229] Synthesis of PAI Salts

[0230] A solution of an appropriate acid (1 mol-eq.) in 2-propanol wasadded to a solution of PAI (1 mol-eq.) while stirring in 2-propanol (Ar,BHT). The salt formed was filtered, washed with 2-propanol and ether,and dried under low pressure. Yields were between 70 to 90%. Anexception in preparing PAI acetate involved using ether as the solvent.

[0231] Analytical Methods

[0232] The chromatographic separations were carried out using aLichrosphere 60 RP select B 5 μ 125×4 mm (Merck) column, an HPLC (JascoBIP-1) equipped with a L-4200 UV-Vis detector (Merck-Hitachi) set to 210nm, and a D-2500 chromatointegrator (Merck-Hitachi). The eluent anddiluent consisted of 80% distilled water/20% acetonitrile (HPLC grade),and 0.07 M perchloric acid adjusted to pH 2.5 with aqueous ammonia. Theflow rate used was 1 ml/min, the appropriate PAI salt solutionconcentration was 250 μg/ml, and 20 μl of the solution were injectedonto the chromatographic system.

[0233] The melting range was measured with an automatic apparatus(Mettler FP 80) and thermo-gravimetric analysis was performed on aMettler TA 3000 system at a rate of 10° C./min in the applicable range.Solubility was determined by an appropriate dilution of the supernatantfrom a saturated PAI salt water solution and measured in a UVIKON 941(Kontron) UV-Vis spectrophotometer. The salt form (mono- or di-salt) wasobtained by elemental analysis using standard equipment for C, H, N andS determination. The pH was measured in a 1% aqueous solution of the PAIsalts.

[0234] Results

[0235] The characterization of the various salts are summarized in Table8. TABLE 8 PHYSICOCHEMICAL PROPERTIES OF PAI SALTS Melting PAI-saltSolubility range Salt m.w. pH mg/ml (° C.) % Wt. loss form tartarate 5.533 176.2-177.3 LT 0.1 di 492 mesylate 4.3 635 156.8-157.6 0.1 mono 267maleate 4.0 NLT 1000  87.2-87.8 0.1 mono 287 sulphate 3.9 485159.4-161.1 3.2 di 440 chloride 4.2 238 177.0-180.0 LT 0.5 mono 207tosylate 4.4 60-70 129.3-129.9 LT 0.1 mono 343 fumarate 3.5 95125.4-126.2 0.2 mono 287 phosphate 7.0 NLT 720 109.5-110.4 n.a. n.a.n.a. esylate 2.4 NLT 300 n.a. n.a. mono 279 acetate 6.1 NLT 720 69.2-69.7 0.4 mono 231

[0236] Comparative stability studies were carried out under sets ofseveral accelerating conditions: I) heating at 80° C. for 72, 96 or 144hours; and II) reflux in isopropanol for 30 hours. The degradationproducts developed were measured by HPLC and confirmed by TLC. Theresults are presented in Table 9 with the relative retention time(relative to the PAI peak; RRT) as an area percentage relative to totalintegrated peak area. TABLE 9 DEGRADATION PRODUCTS DEVELOPED IN PAISALTS UNDER SHORT TERM CONDITIONS Reflux in 80 C/72 h 80 C/144 hiPrOH/30 h Salt RRT^(a) %^(b) RRT % RRT % Sulfate ND^(c) ND ND ND 0.470.22 0.60 0.72 phosphate 0.60 0.22 0.60 0.57 0.60 2.62 0.74 0.21 1.840.20 1.98 0.73 chloride ND ND ND ND 2.23 0.71 mesylate ND ND ND ND 0.600.08 maleate 0.60 0.41 n.a. 0.60 2.17 1.27 0.50 0.65 1.35 1.48 0.33 1.290.59 1.81 0.10 1.42 1.30 3.07 1.44 1.50 0.16 4.16 0.10 1.83 0.18 4.847.76 1.98 0.23 4.09 0.65 acetate 0.44 0.10 n.a. 0.60 6.74 0.60 2.56 0.740.35 0.73 0.13 1.76 0.33 1.29 0.71 1.84 0.16 1.55 1.06 1.99 4.17 1.7521.85 3.60 0.27 1.96 3.33 2.15 0.08 2.32 0.15 2.83 0.15 3.54 1.82esylate^(d) ND ND 0.85 0.26 ND ND 1.96 0.31

[0237] The salts were submitted to visual inspection of color and form.The findings are shown in Table 10. TABLE 10 APPEARANCE OF PAI SALTSUNDER DESTRUCTIVE CONDITIONS reflux in Salt 80° C./72 h 80° C./96 h 80°C./144 h iPrOH/30 h sulfate off white n.a. off white brown powder powderpowder phosphate brownish n.a. brown brown powder powder powder chloridewhite n.a. white off white powder powder powder mesylate white n.a.white white powder powder powder maleate brown brown n.a. brown meltedmelted esylate brownish n.a. dark brown dark brown melted melted melted

[0238] These studies show that sulphate, esylate and mesylate possesssignificant advantages relative to the other salts due to goodsolubility and chemical stability. Of these three salts, mesylate ispreferable due to its excellent stability even under destructiveconditions.

EXAMPLE 32 Reversal of Haloperidol-induced Catalepsy in Mice

[0239] Male, ICR mice 25-30 g each, were pretreated with either of thefollowing drugs: Saline, (R)-PAI mesylate, or racemic-PAI mesylate. Alldrugs were administered i.p. in a volume of 0.2 mL. Two hours later,haloperidol was injected s.c. at a dose of 6 mg/kg in a volume of0.1-0.2 mL. Motor coordination tests were made at 3 hours after givinghaloperidol, that is, 5 hours after administering the presumedprotective drugs.

[0240] Motor coordination tests and rigidity were quantified accordingto three different parameters: (a) ability to walk the length of ahorizontal rod, 80 cm-long; (b) ability to climb down, face down, avertical rod, 80 cm-long; (c) duration of immobility in an unnaturalsitting posture whereby the abdomen of the mouse is pressed against a“wall.” Full performance as in haloperidol-untreated mice is given thescore of 4 in each test, or a total of 12 in all tests. Poor performanceis given a score from 1 to 3. A key to score ratings is given in Table9A. The effects of the various agents in antagonizinghaloperidol-induced catalepsy are given in Table 11. At three hoursafter haloperidol, (R)-PAI mesylate conferred protection againsthaloueridol at 5-15 mg/kg, reaching a peak after effect at 7.5-10 mg/kg(activity score—94% of saline control). Racemic PAI mesylate conferredpartial protection in the range of 7.5-15 mg/kg, and was not active at 5mg/kg. From FIG. 17, it can been seen that the dose-effect profile ofeither (R)-PAI mesylate or racemic PAI is such that an increase in dosebeyond 10 mg/kg entails a decrease in effect, but that the racemicmixture is less potent throughout. This means that racemic PAI mesylateat twice the dose of (R)-PAI mesylate will always be less active thanthe (R) enantiomer.

[0241] Reversal of α-MpT-induced Hypokinesia in Rats

[0242] The drug α-MpT is assumed to inhibit the formation of L-DOPA fromtyrosine, and consequently the formation of dopamine itself. Lack of CNSdopamine is expressed as hypoactivity. Six month-old male Wistar rats(from Harlan Orkack, UK) were pretreated with saline, (R)-PAI Mesylateor Rac PAI Mesylate, at the indicated doses. Two hours later theyreceived i.p. α-MpT at a dose of 100 mg/kg in 0.3-0.5 mL. Controlsreceived saline. Following this, motor activity was recorded in acomputerized activity cage for the duration of 10 hours. The results aregiven in Table 12 and FIG. 18. At 2 mg/kg, (R)-PAI Mesylate restored thelevel of activity to about 90% of the saline-treated rats, but Rac PAIMesylate was not active. In either case, the profile of the dose-effectcurve was bell-shaped, suggesting a decrease in effect with an increasein dose beyond a peak of 2-5 mg/kg. At 5 mg/kg Rac PAI Mesylate couldnot elicit a level of activity comparable to that of (R)-PAI Mesylate at2 mg/kg.

[0243] From these measurements, (R)-PAI Mesylate and Rac PAI-Mesylate donot share a similar pattern of activity in the restoration ofnormokinesia in haloperidol-treated mice and α-Mpt-treated rats. At alldoses studied, (R)-PAI Mesylate is always more potent that Rac PAIMesylate at the corresponding dose. Also, peak activity of Rac PAIMesylate is always lower than peak activity of (R)-PAI Mesylate. Thus,Rac PAI Mesylate at a given dose is always less effective than (R)-PAIMesylate at half the same dose. Doubling the dose of Rac PAI Mesylatewith respect to (R)-PAI Mesylate does not produce an effect equivalentto that of (R)-PAI Mesylate.

[0244] Pharmacologically, Rac PAI Mesylate cannot be considered asconsisting of 50% active ingredient which is (R)-PAI Mesylate and 50%inert material as diluent. The presence of (S)-PAI in Rac PAI Mesylatehas an adverse effect on the activity of (R)-PAI, resulting in a morethan two-fold decrease in potency. The decrease may be due to a directadverse effect of (S)-PAI on behavioral parameters. TABLE 11 REVERSAL OFHALOPERIDOL-INDUCED CATALEPSY IN MICE WITH (R)-PAI MESYLATE AND RACEMICMESYLATE Mice received each of the test drugs i.p. at the indicateddoses. Two hours later they received haloperidol as described in thetext. The doses shown are for the free base. (R)-PAI Mesylate Rac PAIMesylate Dose, % of % of mg/kg Score + SE n control Score + SE n control 1.8  7.2 ± 1 6 60 7.0 ± 0.6 6 59  3.0  6.4 ± 0.5 6 60 5.9 ± 0.7 6 49 5.0  8.7 ± 0.9* 6 73 6.4 ± 0.4 6 53  7.5 11.0 ± 0.4*** 5 92 9.4 ± 0.8++6 78 10 11.3 ± 0.3*** 6 94 9.2 ± 0.6*** 6 77 15 10.8 ± 0.5*** 5 90 8.8 ±0.8* 6 73 Control   12 ± 0 12 100 saline Halo-  6.6 ± 0.3 16 59 peridolalone

[0245] TABLE 11A KEY TO SCORE RATING OF HALOPERIDOL-INDUCED CATALEPSY INMICE AND ITS REVERSAL BY VARIOUS AGENTS Vertical Rod: Unable to grasprod with limbs 1 Able to grasp but slips down 2 Able to grasp, partlyslips, partly climbs down 3 Able to grasp, climbs down using all limbs 4Horizontal Rod: Unable to grasp, falls off rod 1 Able to grasp, unableto walk on rod more than 2 paces 2 Able to grasp, walks half-length ofrod 3 Able to grasp, walks full length of rod 4 Immobility SittingAgainst Wall: Immobility >5 min 1 Immobility 3-5 min 2 Immobility 1-3min 3 Immobility 0.1 min 4

[0246] Fractional scores are assigned, such as 2.5, when behavior fallsbetween two categories, as between 2 and 3. TABLE 12 RESTORATION OFMOTOR ACTIVITY IN RATS TREATED WITH α-METHYL-p-TYROSINE (α-MpT) AT 100mg/kg i.p. Rats received the test drugs i.p. at the indicated doses.After two hours they received α-MpT and were immediately placed inactivity cages. Total motor activity was automatically recorded for 10hours, as described in the text. (R)-PAI Mesylate Rac PAI Mesylate Dose,% of % of mg/kg Score + SE n control Score + SE n control 2  14,132** ±7 89   9,035 ± 6 57 1457 829 5   12,893* ± 7 81 10,926* ± 8 69 1,869 8207.5     6,679 ± 4 42   9,698 ± 4 61 414 557 Control    15,862 ± 5 100saline 1,424 α-Mpt  8,108*** ± 6 51 alone 810

EXAMPLE 33 The Effects of (R)-PAI Mesylate Following Closed Head Injuryin Rats

[0247] Methods

[0248] 1. Induction of Trauma

[0249] Head trauma was induced in male rats under ether anesthesia by awell calibrated weight-drop device that falls over the exposed skull,covering the left cerebral hemisphere, 1-2 mm lateral to the midline, inthe midcoronal plane.

[0250] 2. Evaluation of Motor Function

[0251] One hour after induction of trauma, the rats were tested by a setof criteria which evaluated their neurologic outcome (the criteriadescribed by Shohami, et al., J. Neurotrauma, 10, 113 (1993)). Thesecriteria, referred to as the Neurological Severity Score (NSS), consistof a series of reflexes and motor functions. Points are given based ondeficits in these criteria. At 24 h the rats were re-evaluated.

[0252] 3. Evaluation of Brain Edema

[0253] The brains were removed after the second evaluation of motorfunction (24 h). A piece of tissue (−20 mg) was weighed to yield wetweight (WW). After drying in a desiccator oven for 24 h at 95° C., itwas reweighed to yield dry weight (DW). Water percentage in the tissuewas calculated as (WW-DW)×100/WW.

[0254] 4. Drug Treatment

[0255] (R)-PAI Mesylate was dissolved in water. The rats were injectedintraperitoneally at a dose of 0.1 mg/kg, 0, 4, 8 and 12 h afterinduction of head trauma. Control rats were treated with water at thesame times.

[0256] Results

[0257] The NSS, which measures the “clinical” status of the rats, wasalmost identical in the treated and nontreated groups at 1 hour afterthe head injury, but significantly lower at 24 hours in the (R)-PAImesylate-treated rats (Table 13). These results indicate that PAImesylate is effective in improving motor function recovery followingclosed head injury in rats.

[0258] At 24 hours after trauma, a major edema was found in thehemisphere (85.4% water in the brain of control rats vs. 78.5% inundamaged brain tissue). PAI mesylate was effective in reducing edema asverified by its effect on the percent of water.

[0259] In conclusion, the results reported herein demonstrate that(R)-PAI mesylate has neuroprotective properties in a model intended tomimic human nerve injury and to induce trauma to a closed skull. TABLE13 NSS Δ NSS % H₂O 1 h 24 h (1 h-24 h) in the brain control 15.6 12.34.3 ± 0.5 85.4 ± 0.4 (n = 6) (R)-PAI 16.7 10.2 6.5 ± 0.7* 82.1 ± 0.6**Mesylate (n = 6)

EXAMPLE 34 Effects of PAI Mesylate on Prevention of NMDA Induced CellDeath of Cerebellum Cell Cultures

[0260] Results of in vitro Assays

[0261] Procedures: Cultures of mechanically dissociated neonatal ratcerebellum. The cerebella are dissected aseptically from 6 or 7-day-oldrat pups and placed in a 15 ml sterile plastic conic tube containing 3ml of enriched medium (the medium is made up of Dulbecco's modifiedEagle's medium (DMEM) with high glucose concentration (1 g/l), 2 mM(v/v) L-glutamine, antibiotic antimitotic mixture, and enriched with 15%(v/v) heat-inactivated fetal calf serum). The cerebella are thendissociated after 20-25 passages through a sterile 13 gauge, 10 cm longstainless steel needle attached to a 5 ml syringe with an inserted 45 μmpore size nylon sieve. The dissociated cells are centrifuged at 200 gfor 5 minutes, the supernatant discarded and the cells resuspended inenriched medium. The cell viability is determined by the trypan blueexclusion test. The cells are then plated at a density of 200/mm² onpoly-L-lysine-coated surfaces (Poly-L-lysine-coated glass coverslips areprepared at least 1 hour before plating, by immersing in a steriledistilled water solution containing 15 μg/ml poly-L-lysine, and justbefore use, washing with sterile water and drying), covered withenriched medium, and incubated at 37° C. in an atmosphere of 5% CO₂ inair and 100% humidity. After 4 days in culture, the media are replacedwith media containing the desired test compounds. Experiments are donein duplicate and repeated 2 or 3 times. After determining the testcompound toxic dose-response, four groups are compared: (I) control(enriched medium alone), (II) test compound (one subgroup for eachconcentration (2 concentrations are tested)), (III) N-methyl-D-aspartate(NMDA, exposure to a concentration of 1 mM for 3 h) as the cytotoxicchallenge, (IV) test compound plus NMDA (one subgroup for each of the 2concentrations of test compounds), (V) control group to test the effectof solvent (in which the test compound is dissolved), and (VI) anadditional “positive control” group of spermine (0.01 μM dissolved inculture medium) plus NMDA. Nerve cell survival is evaluated by phasecontrast microscopy and trypan blue staining after 24 h.

[0262] Results

[0263] It is well established that glutamic acid (Glu) possessesneurotoxic properties which are expressed in several neurologicaldisorders including epilepsy and stroke, and most likely also in brainneurodegenerative diseases such as Parkinson's disease, Alzheimer'sdisease and traumatic brain injury. The neurotoxic effects of Glu aremediated by membrane bound Glu receptors, such as N-methyl-D-asparate(NMDA) receptors.

[0264] The results, as shown in Table 14, demonstrate that 10 μM of(R)-PAI mesylate increased the survival of cerebellum cells by 27percent following 1 μM NMDA exposure. These in vitro results support thein vivo effects of (R)-PAI mesylate presented in Examples 33 and 35,indicating that this drug has neuroprotective properties againstneurotoxic concentration of NMDA. TABLE 14 NEUROPROTECTIVE EFFECT OF(R)-PAI MESYLATE ON PREVENTION OF NMDA-INDUCED CELL DEATH OF CEREBELLUMCELLS Experimental Group Surviving Cells Percent Protection CerebellarCultures (Toxicity TD₂₅ = 30 μM; TD₅₀ = 85 μM; TD₁₀₀ = 320 μM) Control100 Solvent 97 NMDA 10 Solvent + NMDA 10 0 Compound + NMDA: 1) 0.01 μM +NMDA: 12 2 2) 1.00 μM + NMDA: 22 12 3) 10.00 μM + NMDA: 37 27 Spermine +NMDA 75 65

[0265] Values, expressed as the percent of untreated controls, representthe average of 2 experiments run in duplicate for culture experiments,and the mean±SEM of 4 animals for ischemia. The percent protection valueis the effect of the test compound after subtraction of the solventeffect.

EXAMPLE 35 Effects of (R)-PAI Mesylate After Traded Crush of the RatOptic Nerve

[0266] Neuroprotective effects of (R)-PAI Mesylate were determined forapplication immediately after crush injury of the optic nerve in theadult rat. Short-term effects were measured metabolically, and long-termeffects electrophysiologically.

[0267] Methods

[0268] 1. Metabolic Measurements

[0269] a) General. The method is described by Yoles, et al.,Investigative Ophthalmology & Visual Science, 33, 3586-91 (1992). Atshort terms, metabolic measurements were monitored in terms of themitochondrial NADH/NAD ratio, which depends on the activity of theelectron transport system, and thus indicate levels of energyproduction. Changes in ability of the nerve to produce energy as aconsequence of injury were determined by comparing NADH levels inresponse to artificial transient anoxic insult before and after theinjury.

[0270] b) Surface fluorometry—reflectometry. Monitoring of theintramitochondrial NADH redox state is based on the fact that NADH,unlike the oxidized form NAD, fluoresces when illuminated at 450 nm. Aflexible Y-shaped bundle of optic fibers (light guide) was used totransmit the light to and from the optic nerve. The light emitted fromthe nerve was measured at two wavelengths: 366 nm (reflected light) and450 nm (fluorescent light). Changes in the reflected light werecorrelated with changes in tissue absorption caused by hemodynamiceffects and with movements of the optic nerve secondary to alterationsin arterial blood pressure and nerve volume. The fluorescencemeasurements were found to be adequately corrected for NADH redox statemeasurements by subtraction of the reflected light (366 nm) from thefluorescent light (1:1 ratio) to obtain the corrected fluorescencesignal.

[0271] c) Animal preparation. Animal utilization was in accord with theARVO Resolution on the use of animals in research. Male Sprague-Dawley(SPD) rats weighing 300-400 g were anesthetized with sodiumpentobarbitone (50 mg/kg intraperitoneally). With the animal's head heldin place by a head holder, a lateral canthotomy was performed under abinocular operating microscope and the conjuctiva was incised lateral tothe cornea. After separation of the retractor bulbi muscles, the opticnerve was identified and a length of 3-3.5 mm was exposed near theeyeball by blunt dissection. The dura was left intact and care was takennot to injure the nerve. A special light-guide holder was implantedaround the optic nerve in such a way that the light guide was located onthe surface of the optic nerve 1 mm distal to the injury site. Animals,while still anesthetized, were allowed to recover for 30 minutes fromthe surgical procedures and were then exposed to anoxic conditions. Ananoxic state was achieved by having the rat breathe in an atmosphere of100% nitrogen for 2 minutes, after which time it was returned to air. Inorder to evaluate the metabolic activity of the optic nerve, therelative changes in reflected and fluorescent light intensities inresponse to anoxia were measured before and after crush injury.

[0272] d) Experimental protocol for crush injury and metabolicmeasurements. With the aid of calibrated cross-section forceps, amoderate crush injury was inflicted on the nerve between the eye and thelight guide holder at a pressure corresponding to 120 g for 30 sec.Immediately after injury, animals received intraperitoneal injections ofwater with and without (R)-PAI Mesylate (2 mg/kg). To assess theactivity of the energy production system, NADH response to 2 minutes ofanoxia was measured in all animals prior to injury, 30 minutes afterinjury, and thereafter at hourly intervals up to 4 hours (see FIG. 19).

[0273] 2. Electrophysiological Measurements. This method is described byAssia, et al., Brain Res., 476, 205-212 (1989). Animal preparation andoptic nerve injury were preferred as in the metabolic studies.Immediately after injury, animals received a single injection of waterwith or without (R)-PAI Mesylate (0.5 mg/kg). Fourteen days after injuryand treatment, the optic nerves were excised and measuredelectrophysiologically. Prior to removal of optic nerves forelectrophysiological measurement, the rats were deeply anesthetized with70 mg/kg pentobarbitone. The skin was removed from the skull and theoptic nerves were detached from the eyeballs. Subtotal decapitation wasperformed and the skull was opened with a rongeur. The cerebrum wasdisplaced laterally, exposing the intracranial portion of the opticnerve. Dissection was at the level of the nerve, which was transferredto vials containing fresh salt solution consisting of NaCl (126 mM), KCl(3 mM), NaH₂PO₄ (1.25 mM), NaHCO₃ (26 mM), MgSO₄ (2 mM), CaCl₂ (2 mM),and D-glucose (10 mM), and aerated with 95% O₂ and 5% CO₂ at roomtemperature. The nerves were kept in this solution, in which electricalactivity remained stable for at least 3-4 hours. After 0.5 hours ofrecovery at room temperature, electrophysiological recordings wereobtained from the nerve distal to the crush lesion. The nerve ends werethen connected to two suction Ag—AgCl electrodes immersed in a bathingsolution at 37° C. A stimulating pulse was applied through the electrodeat the proximal end and the action potential was recorded by the distalelectrode. A Grass SD9 stimulator was used for supramaximal electricalstimulation (0.5 pps). The measured signal was transmitted to a MedelecPA36 preamplifier and then to an electromyograph (Medelec MS7, AA7Tamplifier). The solution, stimulator and amplifier had a common ground.The maximum amplitude of eight averaged compound action potentials(CAPs) was recorded and photographed with a Polaroid camera. The CAPvalues measured in contralateral uninjured nerves served as a reference.

[0274] Results

[0275] The results demonstrate that (R)-PAI Mesylate applied immediatelyafter optic nerve injury blocked the injury-induced reduction in energyproduction. (R)-PAI Mesylate also has a long-term effect measured byelectrophysiological monitoring.

[0276] The CAP (compound action potentials) amplitude is directlycorrelated with the number of conducting fibers in the tested segment ofthe nerve.

[0277] (R)-PAI Mesylate significantly attenuated the injury-induced lossof activity in the distal segment of the injured nerve, indicating that(R)-PAI Mesylate is a neuroprotective agent or at least slows downdegeneration. TABLE 15 Electrophysiological Measurements CAP amplitude(μV) Group (Mean ± Std. Error.) Vehicle  441 ± 95   N = 13 (R) - PAI2104 ± 313* Mesylate N = 7

EXAMPLE 36 Comparison of Anticonvulsive Properties of R-PAI and S-PAISalts

[0278] Both (R)-PAI and (S)-PAI HCl salts have significantanticonvulsant activities. In mice (i.p. administration) in the maximalelectroshock test (MES test), (S)-PAI HCl has greater anticonvulsantactivity (ED₅₀=57 mg/kg) than (R)-PAI HCl (ED₅₀=79 mg/kg). Analogousresults were observed in rats (p.o. administration). Four out of fourrats were protected from seizures in the MES test when 50 mg/kg of(S)-PAI HCl was administered, whereas three out of four mice wereprotected after the same dose of (R)-PAI HCl. With respect to efficacyfor Parkinson's disease, the enhanced anticonvulsant activity is adetrimental side effect. The same trend occurs with the mesylate salts.(S)-PAI Mesylate has greater anticonvulsant activity than (R)-PAIMesylate in the MES test. At doses of 100 mg/kg, (S)-PAI Mesylateprotected three out of three mice, whereas only one out of three micewas protected with (R)-PAI Mesylate.

[0279] The MES test is a classical model to indicate efficacy forpartial and generalized seizures in humans. The agents'mechanism ofaction is via their ability to prevent the spread of seizures. Someagents, however, that prevent seizure spread have the side effect oflowering seizure threshold. These agents therefore have bothproconvulsive and anticonvulsive side effects.

[0280] Results herein show that (S)-PAI Mesylate has proconvulsiveactivity. In the Timed Intravenous Infusion of Metrazol test, 141 mg/kgof (S)-PAI Mesylate reduces the time, and therefore the amount ofMetrazol, required to induce the appearance of both the first focalseizure and the onset of clonus. Other agents that are classically usedfor partial and generalized seizures, such as phenytoin andcarbamazepine, do not show this effect. (H. J. Kupferberg, Epilepsia,30, s51-s56 (1989)). Likewise, (S)-PAI Mesylate showed a significantlyhigher acute neurotoxicity than (R)-PAI Mesylate. At 300 mg/kg, (R)-PAIMesylate did not show any neurotoxicity with mice in the rotorod ataxiatest. With (S)-PAI Mesylate, four out of four mice showed neurotoxicityand spasticity.

[0281] Methods

[0282] TD₅₀ (median toxic dose). This test measures neurologicaldeficits by the rotorod ataxia test. A mouse is placed on a knurled rodrotating at 6 rpm. It is then determined whether a mouse has the abilityto maintain its equilibrium and stay on the rod for one minute in eachof three trials.

[0283] Timed Intravenous Infusion of Metrazol Test. This test measuresthe minimal seizure threshold of each animal. Metrazol is infused at0.185 mg/ml into the tail veins of mice. The time is then recorded (sec)from the start of infusion until the appearance of the first twitch(first focal seizure) and onset of clonus (clonic seizure).Proconvulsants require less Metrazol to produce these symptoms andtherefore show endpoints at a shorter period of time.

EXAMPLE 37 Peripheral Effects of (R)-PAI and (S)-PAI on theContractility of Intestinal Smooth Muscle Preparations

[0284] Peripheral effects of the hydrochloride salts of the enantiomersof PAI were determined in isolated rabbit or guinea-pig small intestine.These observations provide useful information on their relativeperipheral side effects in humans. The first point of contact of thesubject with an orally administered drug is the gastrointestinal tractwhere concentrations of the drug are much higher than after absorptionand distribution. In the case of PAI hydrochloride (MW=208), a 10 mgoral dose contained in a liquid volume of about 100 ml would beequivalent to a concentration of about 0.5 mM. In contrast, thetherapeutic plasma concentration of (R)-PAI hydrochloride is in thenanomolar range.

[0285] The effect of the enantiomers of PAI in the isolated rabbitjejunum and the guinea-pig ileum were determined so as to find outwhether the intake of (S)-PAI together with (R)-PAI (as found in racemicPAI) would produce side effects absent in the administration of pure(R)-PAI. (R)-PAI is the preferred enantiomer for the inhibition ox MAO-Bin the brain, in view of its potency and high selectivity towards thisform of the enzyme. (S)-PAI is much less potent than (R)-PAI in thisrespect and is also not selective toward MAO-B. In principle, itspresence in PAI racemate might be tolerated or overlooked provided(S)-PAI is inert at the recommended doses of (R)-PAI. The resultsprovided in Tables 16-19 show that (S)-PAI is not an inert substance. Onthe contrary, in the guinea-pig ileum, it is a more potent relaxant than(R)-PAI. Hence its peripheral effects cannot be discounted asnegligible. These data show that there would be fewer peripheral sideeffects in the administration of pure (R)-PAI than in the administrationof racemic PAI containing an equivalent dose of (R)-PAI. TABLE 16TYRAMINE POTENTIATION BY EACH OF THE TWO ENANTIOMERS OF PAI HCl IN RATEJEJUNUM PREPARATION A stretch of rabbit jejunum, mounted in an organbath, displays rhythmic contractions that are inhibited bynorepinephrine but not by tyramine. If however the jejunum is pretreatedwith a monoamine oxidase inhibitor such as PAI, then tyramine causesrelaxation of the spontaneous contractions. The extent of relaxation canbe correlated with the relative potency of the inhibitor. Percent Drugand concentration (μM) relaxation Tyramine alone 40 0 Norepiniphrine0.002 100 (R) PAI alone 0.2-4.0 0 (S) PAI alone 0.2-4.0 0 Tyramine 40after (R) PAI 0.2 67 2 88 40 85-90 after (S) PAI 0.2 0 2 35 40 33-50

[0286] Results

[0287] (S)-PAI is much less potent than (R)-PAI as an inhibitor of brainMAO-B. Therefore, (S)-PAI is not a useful agent for the prevention ofbrain dopamine degradation, but can potentiate the tyramine-evokedrelease of norepinephrine in the small intestine. Its activity in thesmall intestine is an undesirable side effect as it is expected toincrease the absorption and action of undegraded tyramine. Thus, (S)-PAIis not an inert substance when used together with (R)-PAI as found inracemic PAI. TABLE 17 ANTAGONISM OF BETHANECHOL-INDUCED CONTRACTIONS OFTHE GUINEA PIG ILEUM PREPARATION IN THE PRESENCE OF 400 μM OF EACH OFTHE TWO ENANTIOMERS OF PAI HCl A stretch of guinea-pig ileum mounted ina physiological solution in an organ bath contracts dose-dependentlywhen treated with bethanechol which is an enzymatically stable analog ofthe natural gastrointestinal neurotransmitter acetylcholine. Thesecontractions are attenuated in the presence of PAI. The data areexpressed in gram-tension. gram-tension Bathenechol (μM) control (R) PAIcontrol (S) PAI) 0.8 0.5 0.2 0.6 0 2 1.5 0.3 2.0 0 4 2.2 0.7 3.0 0 8 4.01.0 3.8 0.6 20 5.6 2.0 3.8 1.2 40 6.2 2.8 3.8 1.7 80 6.2 3.1 3.8 2.6 2006.2 4.3 3.8 2.6

[0288] Results

[0289] (S)-PAI is almost inactive as a MAO-B inhibitor with respect to(R)-PAI, and hence is not effective in preventing the degradation ofbrain dopamine. However, it is more effective than R(PAI) in theprevention of the bethanechol-induced contraction of the smallintestine. Thus (S)-PAI is not an inert substance when used with R(PAI)as found in racemic PAI. TABLE 18 ANTAGONISM OF THE HISTAMINE-INDUCEDCONTRACTIONS OF THE GUINEA-PIG ILEUM PREPARATION BY EACH OF THE TWOENANTIOMERS OF PAI HCl A fixed dose of histamine (40 nM) causes asustained contraction of a stretch of guinea-pig ileum mounted inphysiological solution in an organ bath. Incremental addition of each ofthe two enantiomers of PAI HCl causes a dose-dependent relaxation of themuscle. Results are expressed as percent relaxation with respect to thebase- line before addition of histamine, which is taken as 100%relaxation. PAI concentration Percent relaxation μM (R) PAI (S) PAI 2 011 4 0 15 10 0 30 20 20 30 31 33 40 37 36 100 81 71 200 90 300 92 400100 98 700 100 1000 100

[0290] Results

[0291] (S)-PAI is inactive with respect to (R)-PAI as a MAO-B inhibitorin the brain, and hence useless for preventing the degradation of braindopamine, but is more active than the (R) isomer in causing relaxationof intestinal smooth muscle. Thus, (S)-PAI is not an inert substancewhen taken together with the (R)isomer as found in racemic PAI. TABLE 19ANTAGONISM OF THE BETHANECHOL-INDUCED CONTRACTIONS OF THE GUINEA-PIGILEUM PREPARATION BY EACH OF THE TWO ENANTIOMERS OF PAI HCl A fixed doseof bethanechol (0.8 μM) causes a sustained contraction of a stretch ofguinea-pig ileum mounted in physiological solution in an organ bath.Incremental addition of each of the two enantiomers of PAI HCl causes adose-dependent relaxation of the preparation. Results are expressed aspercent relaxation with respect to the base- line before addition ofhistamine, which is taken as 100% relaxation. PAI concentration Percentrelaxation μM (R) PAI (S) PAI 20  25 40-50 60 25-50 60-70 100 50-70 100300 100 100

[0292] Results

[0293] (S)-PAI is inactive with respect to (R)-PAI as a MAO-B inhibitorin the brain, and hence useless for the prevention of the gradation ofbrain dopamine, but is more active than the (R) isomer in causingrelaxation of intestinal smooth muscle. Thus, (S)-PAI is not an inertsubstance when taken together with the (R) isomer as found in racemicPAI.

EXAMPLE 38 Some Effects of [R](+)PAI Mesylate in Middle Cerebral ArteryOcclusion in the Rat as a Model for Stroke

[0294] Methods

[0295] 1.1. Middle Cerebral Artery Occlusion (MCAO) in the Rat.

[0296] A modification of the procedure described by Tamura et al (1981)was used. Male Wistar rats (Olac England-Jerusalem) 300-400 g each wereanesthetized with a solution of Equitesine administered i.p. at a doseof 3 mL/kg. Equitesine consists of 13.5 mL sodium pentothal solution (60mg/mL), 3.5 g chloral hydrate, 1.75 g MgSO₄, 33 mL propylene glycol, 8.3mL absolute alcohol made up to 83 mL with distilled water. Surgery wasperformed with the use of a high magnification operating microscope,model SMZ-2B, type 102 (Nikon, Japan). In order to expose the leftmiddle cerebral artery, a cut was made in the temporal muscle. The tipof the coronoid process of mandible was excised as well and removed witha fine rongeur. Craniectomy was made with a dental drill at the junctionbetween the median wall and the roof of the inferotemporal fossa. Thedura matter was opened carefully using a 27 gauge needle. The MCA waspermanently occluded by microbipolar coagulation at low power setting,beginning 2-3 mm medial to the olfactory tract between its corzicalbranch to the rhinal cortex and the laterate striate arteries.

[0297] After coagulation, the MCA was severed with microscissors anddivided to ensure complete occlusion. Following this, the temporalismuscle was sutured and laid over the craniectomy site. The skin wasclosed with a running 3-0 silk suture. A sham craniectomy operation wasperformed on a parallel group of rats, but without cauterization of theMCA. During the entire surgical operation (20-25 min) in either group,body temperature was maintained at 37 to 38° C. by means of abody-temperature regulator (Kyoristsu, Japan) consisting of aself-regulating heating pad connected to a rectal thermistor. At 24hours post surgery a neurological score was taken in order to assess theseverity of the injury in the drug-treated rats with respect to theiruntreated controls. At 48 hours, the animals were anesthetized withEquitesine and the severity of the injury was visualized by an MRIprocedure. The volume of brain tissue incurring damage followingischemia was determined.

[0298] 1.2. Drug Administration

[0299] [R](+)PAI Mesylate was administered as an i.p. injection in0.3-0.4 mL distilled water, according to the following schedule:

[0300] 1 mg/kg immediately after surgery.

[0301] 0.5 mg/kg 2 hours after surgery

[0302] 1 mg/kg 20-24 hours after surgery

[0303] 1.3. MRI Scan of Ischaemic Brain Lesion

[0304] All experiments were performed using a 4.7 T BIOSPEC system(BRUKER) (See T. Back, et al., “Diffusion Nuclear Magnetic ResnonanceImaging in Experimental Stroke: Correlation with Cerebral Metabolites,”Stroke (February 1994) 25: 494-500). Forty-eight hours after MCAO orsham operation, every animal was subjected to a fast multislices T1weighted imaging (TR/TE), (500/25) for positioning. Then multislicesT2-weighted images (3000/80) were acquired (5 contiguous slices, 3 mmthick).

[0305] The size and severity of the infarcted area was estimated usingthe hyperintensity observed in the T2 weighted MRI at 48 hourspost-occlusion or post sham-operation. The following MRI parameters weredetermined for each group of rats:

[0306] c. Ischemic area (in mm²)

[0307] d. Area of the ischemic hemisphere (in mm²)

[0308] e. Area of the unaffected hemisphere (in mm²)

[0309] The use of contiguous slices allows the conversion of area unitsinto volume units by simply multiplying the area value by the slicethickness.

[0310] 1.4. Neurological Score

[0311] The neurological score consists of the sum total of a series ofratings assigned to the performance of specific locomotor activities ina given rat. The scale runs from 0 (fully normal rats) to 13 (fullyincapacitated rats). Most parameters are rated as either 0 (normal), or1 (incapacitated); others are graded. The following tests were used inthe present study:

[0312] General Observational Tests: Hypoactivity; Sedation; Piloerection

[0313] Motor reflex. Rats were lifted by the tail about 15 cm above thefloor. Normal rats assume a posture in which they extend both forelimbstowards the floor and spread the hind limbs to the sides in atrapeze-like manner. MCAO when severe causes consistent flexion of thecontralateral limb.

[0314] Motor ability. This is seen as the ability to grasp a rod 1 cm indiameter by the contralateral limb for 5-15 sec when the rat is lefthanging on the rod through the arm pit.

[0315] Motor coordination. Normal rats are able to walk up and down abeam 5 cm wide placed at a moderate slant. Failure to walk the beam ineither direction reveals some motor incoordination, lack of balance andlimb weakness.

[0316] Gait. Ability to restore normal position to either hindcontralateral limb when intentionally displaced while on a narrow beam.

[0317] Balance. Ability to grasp and balance on a narrow beam 2 cm wide.

[0318] Locomotor activity. Total movements over a period of 15 min in anautomated activity cage.

[0319] Ratings assigned to each of the above parameters are given inTable 20. TABLE 20 Neurological scores assigned to each of 10 parametersof posture and locomotion Parameter Score a. Activity in the home cagenormal = 0 hypoactive = 1 b. Sedation none = 0 marked = 1 c. Piloeretionnone = 0 marked = 1 d. Extension of contralateral forelimb towards good= 0 floor when lifted by tail flexed limb = 1 e. Spread of contralateralhind limb when lifted good = 0 by tail (trapezoid posture) flexed limb =1 f. Grasp rod with contralateral limb for 5-15 sec. good = 0 poor = 1when suspended by the armpit g. Walk on beam 5-cm wide good = 0 poor = 1h. Restoration of contralateral good = 0 hind and or fore limb tooriginal position poor = 1 (one limb) when intentionally 2 (two limbs)displaced i. Grasping and balance on beam good = 0 poor = 1 2-cm wide j.Motor activity with respect to control ≦25% of control 3 (for 15 min inan automated 26-50% of control 2 activity cage 51-75% of control 176-100% of control 0

[0320] 2. Results

[0321] 2.1. Infarct Size

[0322] The results of the MRI study are summarized in Table 21 and FIG.20. The infarct size was significantly smaller in [R](+)PAIMesylate-treated rats (n=9) than in untreated rats (n=10). In theformer, the infarct size was about 60% of that in the untreated animals.TABLE 21 Ischaemic brain lesion evaluation by MRI T2-SCAN - 48 hrsfollowing MCA-Occlusion and [R] (+) PAI Mesylate treatment in WistarRats. MCA-O + MCA-O [R] (+) PAI Mesylate* Infarct size Infarct sizeAnimal No. (mm³) Animal No. (mm³)  1 252 1 94.4  2 272 2 139  3 314 3240  4 273 4 137  5 201 5 137  6 221 6 174  7 358 7 164  8 265 8 171  9341 9 215 10 236 MEAN ± SD 273.3 ± 50.9 MEAN ± SD 163.5 ± 43.9 t =5.0475 f = 17 p < 0.001 [R] (+) PAI Mesylate reduces infarct size by 40%significantly *[R] (+) PAI Mesylate administered: Time afterMCA-Occlusion: 0 −1.0 mg/kg ip;  2 hrs −0.5 mg/kg ip; 24 hrs −1.0 mg/kgip.

[0323] 2.2. Neurological Score

[0324] The neurological score in five [R](+)PAI Mesylate treated ratsand six untreated rats were determined by a blinded observer. Theresults are given in Table 22 where they are compared with the infarctsize in each animal as determined by the MRI test, and also in FIG. 21.It can be seen that those animals with the least neurological scoreswere those treated with [R](+)PAI Mesylate. The neurological score wasreduced by 54% and the infarct size by 36% in [R](+)PAI Mesylate-treatedMCAO rats as compared to untreated ones. TABLE 22 Neurological score ofRats submitted to MCA-Occlusion and [R] ( + ) PAI Mesylate treatmentwith relation to their ischaemic infarct size. MCA-O MCA-O + [R] (+) PAIMesylate*** Infarct Infarct Animal Neurological* size** AnimalNeurological* size** No. Score (mm³) No. Score (mm³) 1 5.0 201 1 1.0 1372 5.0 221 2 2.0 174 3 6.0 358 3 4.0 164 4 6.0 265 4 4.0 171 5 8.25 341 52.88 215 6 5.75 236 MEAN + SD 6.0 ± 1.19 270 ± 65 MEAN + SD 2.78 ± 1.3172 ± 28 Neurological Score Infarct Size t = 4.25 t = 3.34 f = 9 f = 9 p< 0.01 p < 0.01

[0325] References for Example 38

[0326] Cechetto D F, Wilson J X, Smith K E, Wolski D, Silver M D,Hachinski V C (1989). Autonomic and myocardial changes in middlecerebral artery occlusion: stroke models in the rat. Brain Res502:296-305.

[0327] Kolb B, Sutherland R J, Whishaw I Q (1983). A comparison of thecontributions of the frontal and parietal association cortex to spatiallocalizations in the rat. Behav. Neurosci. 97:13-27.

[0328] Menzies S A, Hoff J T, Betz A L (1992). Middle cerebral arteryocclusion in rats: A neurological and pathological evaluation of areproducible model. Neurosurgery 31:100-106.

[0329] Sauer D, Allegrini P R, Cosenti A, Pataki A, Amaceker H, Fagg G E(1993). Characterization of the cerebroprotective efficacy of thecompetitive NMDA receptor antagonist CGP40116 in a rat model of focalcerebral ischemia: An in vivo magnetic resonance imaging study. J.Cerebr. Blood Flow and Metabol. 13:595-602.

[0330] Stephanovich C, Editor, Stroke: Animal Models, Pergamon Press,1983

[0331] Tamura A, Graham D I, McCulloch J, Teasdale G H (1981). Focalcerebral ischemia in the rat: 1. Description of technique and earlyneuropathological consequences following MCA occlusion. J. Cereb. BloodFlow and Metab. 1:53-60.

[0332] Teasdale G, Tyson G, Tamura A, Graham D I, McCulloch J. Focalcerebral ischaemia in the rat: Neuropatholgy, local cerebral blood flowand cerebrovascular permeability. In Stroke: Animal Models, StephanovicC, Editor, Pergamon Press 1983, pp.83-97.

[0333] Yamamoto M, Tamura A, Kirino T, Shimitzu M, Sano K (1988).Behavioral changes after focal cerebral ischemia by left middle cerebralartery occlusion in rats. Brain Re. 452:323-328

[0334] Yamori Y et al. (1976). Pathogenic similarity of strokes instroke prone spontaneously hypertensive rats and humans. Stroke 7:46-53.

[0335] Young W, DeCrescito V, Flamm E S, Hadani M, Rappaport H, Cornu P(1986). Tissue Na, K, and Ca changes in regional cerebral ischemia:Their measurement and interpretation. Central Nervous System Trauma,3:215-234.

What is claimed is:
 1. A method of treating brain ischemia or stroke ina subject which comprises administering to the subject an amount ofR(+)-N-propargyl-1-aminoindan or a pharmaceutically acceptable saltthereof effective to treat brain ischemia or stroke in the subject. 2.The method of claim 1, wherein the pharmaceutically acceptable salt ofR(+)-N-propargyl-1-aminoindan is selected from the group consisting of:the mesylate salt; the ethylsulfonate salt; the sulfate salt; and thehydrochloride salt.
 3. The method of claim 2, wherein thepharmaceutically acceptable salt is the mesylate salt ofR(+)-N-propargyl-1-aminoindan.
 4. The method of claim 1, wherein theeffective amount is from about 0.5 milligrams per kilogram body weightof the subject to about 2.5 milligrams per kilogram body weight of thesubject.
 5. The method of claim 1, wherein theR(+)-N-propargyl-1-aminoindan or pharmaceutically acceptable saltthereof is administered intravenously, orally, rectally, transdermally,or parenterally.
 6. The method of claim 1, wherein the subject is humanand the effective amount is from about 0.01 mg to 50.0 mg per day. 7.The method of claim 6, wherein the effective amount is from 0.1 to 10.0mg per day.
 8. The method of claim 6, wherein theR(+)-N-propargyl-1-aminoindan or pharmaceutically acceptable saltthereof is administered intravenously.
 9. The method of claim 1, whereinthe area of the brain ischemia is reduced by about thirty-five percent.10. A method of treating head trauma injury in a subject which comprisesadministering to the subject an amount of R(+)-N-propargyl-1-aminoindanor a pharmaceutically acceptable salt thereof effective to treat headtrauma injury in the subject.
 11. The method of claim 10, wherein thepharmaceutically acceptable salt of R(+)-N-propargyl-1-aminoindan isselected from the group consisting of: the mesylate salt; theethylsulfonate salt; the sulfate salt; and the hydrochloride salt. 12.The method of claim 11, wherein the pharmaceutically acceptable salt isthe mesylate salt of R(+)-N-propargyl-1-aminoindan.
 13. The method ofclaim 10, wherein the effective amount is from about 0.5 milligrams perkilogram body weight of the subject to about 2.5 milligrams per kilogrambody weight of the subject.
 14. The method of claim 10, wherein theR(+)-N-propargyl-1-aminoindan or pharmaceutically acceptable saltthereof is administered orally, rectally, transdermally, orparenterally.
 15. The method of claim 10, wherein the subject is humanand the effective amount is from about 0.01 mg to 50.0 mg per day. 16.The method of claim 15, wherein the effective amount is from 0.1 to 10.0mg per day.
 17. The method of claim 15, wherein theR(+)-N-propargyl-1-aminoindan or pharmaceutically acceptable saltthereof is administered intravenously.
 18. A method of treating spinaltrauma injury in a subject which comprises administering to the subjectan amount of R(+)-N-propargyl-1-aminoindan or a pharmaceuticallyacceptable salt thereof effective to treat spinal trauma injury in thesubject.
 19. The method of claim 18, wherein the pharmaceuticallyacceptable salt of R(+)-N-propargyl-1-aminoindan is selected from thegroup consisting of: the mesylate salt; the ethylsulfonate salt; thesulfate salt; and the hydrochloride salt.
 20. The method of claim 19,wherein the pharmaceutically acceptable salt is the mesylate salt ofR(+)-N-propargyl-1-aminoindan.
 21. The method of claim 18, wherein theeffective amount is from about 0.5 milligrams per kilogram body weightof the subject to about 2.5 milligrams per kilogram body weight of thesubject.
 22. The method of claim 18, wherein theR(+)-N-propargyl-1-aminoindan or pharmaceutically acceptable saltthereof is administered orally, rectally, transdermally, orparenterally.
 23. The method of claim 18, wherein the subject is humanand the effective amount is from about 0.01 mg to 50.0 mg per day. 24.The method of claim 23, wherein the effective amount is from 0.1 to 10.0mg per day.
 25. The method of claim 23, wherein theR(+)-N-propargyl-1-aminoindan or pharmaceutically acceptable saltthereof is administered intravenously.
 26. A method of treatingneurotrauma in a subject which comprises administering to the subject anamount of R(+)-N-propargyl-1-aminoindan or a pharmaceutically acceptablesalt thereof effective to treat neurotrauma in the subject.
 27. Themethod of claim 26, wherein the pharmaceutically acceptable salt ofR(+)-N-propargyl-1-aminoindan is selected from the group consisting of:the mesylate salt; the ethylsulfonate salt; the sulfate salt; and thehydrochloride salt.
 28. The method of claim 27, wherein thepharmaceutically acceptable salt is the mesylate salt ofR(+)-N-propargyl-1-aminoindan.
 29. The method of claim 26, wherein theeffective amount is from about 0.5 milligrams per kilogram body weightof the subject to about 2.5 milligrams per kilogram body weight of thesubject.
 30. The method of claim 26, wherein theR(+)-N-propargyl-1-aminoindan or pharmaceutically acceptable saltthereof is administered orally, rectally, transdermally, orparenterally.
 31. The method of claim 26, wherein the subject is humanand the effective amount is from about 0.01 mg to 50.0 mg per day. 32.The method of claim 31, wherein the effective amount is from 0.1 to 10.0mg per day.
 33. The method of claim 31, wherein theR(+)-N-propargyl-1-aminoindan or pharmaceutically acceptable saltthereof is administered intravenously.
 34. A method of treating asubject afflicted with a neurodegenerative disease which comprisesadministering to the subject an amount of R(+)-N-propargyl-1-aminoindanor a pharmaceutically acceptable salt thereof effective to treat theneurodegenerative disease in the subject.
 35. A method of treating asubject afflicted with a neurotoxic injury which comprises administeringto the subject an amount of R(+)-N-propargyl-1-aminoindan or apharmaceutically acceptable salt thereof effective to treat theneurotoxic injury in the subject.
 36. A method of treating a subjectafflicted with brain ischemia which comprises administering to thesubject an amount of R(+)-N-propargyl-1-aminoindan or a pharmaceuticallyacceptable salt thereof effective to treat brain ischemia in thesubject.
 37. A method of treating a subject afflicted with a head traumainjury which comprises administering to the subject an amount ofR(+)-N-propargyl-1-aminoindan or a pharmaceutically acceptable saltthereof effective to treat the head trauma injury in the subject.
 38. Amethod of treating a subject afflicted with a spinal trauma injury whichcomprises administering to the subject an amount ofR(+)-N-propargyl-1-aminoindan or a pharmaceutically acceptable saltthereof effective to treat the spinal trauma injury in the subject. 39.A method of preventing nerve damage in a subject which comprisesadministering to the subject an amount of R(+)-N-propargyl-1-aminoindanor a pharmaceutically acceptable salt thereof effective to prevent nervedamage in the subject.
 40. The method of claim 39, wherein the nervedamage is structural nerve damage.
 41. The method of claim 39, whereinthe structural nerve damage is optic nerve damage.